Differential roles of HIC-5 isoforms in the regulation of cell death and myotube formation during myogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao Zhengliang; Deblis, Ryan; Glenn, Honor

2007-11-15

Hic-5 is a LIM-Only member of the paxillin superfamily of focal adhesion proteins. It has been shown to regulate a range of biological processes including: senescence, tumorigenesis, steroid hormone action, integrin signaling, differentiation, and apoptosis. To better understand the roles of Hic-5 during development, we initiated a detailed analysis of Hic-5 expression and function in C{sub 2}C{sub 12} myoblasts, a well-established model for myogenesis. We have found that: (1) myoblasts express at least 6 distinct Hic-5 isoforms; (2) the two predominant isoforms, Hic-5{alpha} and Hic-5{beta}, are differentially expressed during myogenesis; (3) any experimentally induced change in Hic-5 expression results inmore » a substantial increase in apoptosis during differentiation; (4) ectopic expression of Hic-5{alpha} is permissive to differentiation while expression of either Hic-5{beta} or antisense Hic-5 blocks myoblast fusion but not chemodifferentiation; (5) Hic-5 localizes to focal adhesions in C{sub 2}C{sub 12} myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (6) alterations in Hic-5 expression interfere with the normal dynamics of laminin expression; and (7) ectopic laminin, but not fibronectin, can rescue the Hic-5-induced blockade of myoblast survival and differentiation. Our data demonstrate differential roles for individual Hic-5 isoforms during myogenesis and support the hypothesis that Hic-5 mediates these effects via integrin signaling.« less

MicroRNA-466 (miR-466) functions as a tumor suppressor and prognostic factor in colorectal cancer (CRC).

PubMed

Tong, Feng; Ying, Youhua; Pan, Haihua; Zhao, Wei; Li, Hongchen; Zhan, Xiaoli

2018-01-17

MicroRNAs (miRNAs) have an important role in the regulation of tumor development and metastasis. In this study, we investigated the clinical and prognostic value as well as biological function of miR-466 in colorectal cancer (CRC). Tumor and adjacent healthy tissues were obtained from 100 patients diagnosed with CRC. miR-466 expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). mRNA and protein levels of cyclin D1, apoptosis regulator BAX (BAX), and matrix metalloproteinase-2 (MMP-2) were analyzed by qRT-PCR and Western blot, respectively, in SW-620 CRC cells transfected with miR-466 mimics or negative control miRNA. Effects of miR-466 on SW-620 cell proliferation, cell cycle and apoptosis, and invasion were investigated using CCK-8 assay, flow cytometry and Transwell assay, respectively. miR-466 expression was significantly downregulated in tumor tissues compared to matched adjacent non-tumor tissues. Low expression of miR-466 was significantly correlated with the tumor size, Tumor Node Metastasis stage, lymph node metastasis, and distant metastasis. The overall survival of CRC patients with low miR-466 expression was significantly shorter compared to high-miR-466 expression group (log-rank test: p = 0.0103). Multivariate analysis revealed that low miR-466 expression was associated with poor prognosis in CRC patients. The ectopic expression of miR-466 suppressed cell proliferation and migration/invasion, as well as induced G0/G1 arrest and apoptosis in SW-620 cells. Moreover, the ectopic expression of miR-466 decreased the expression of cyclin D1 and MMP-2, but increased BAX expression in SW-620 cells. In conclusion, our findings demonstrated that miR-466 functions as a suppressor miRNA in CRC and may be used as a prognostic factor in these patients.

MicroRNA-125b is a novel negative regulator of p53.

PubMed

Le, Minh T N; Teh, Cathleen; Shyh-Chang, Ng; Xie, Huangming; Zhou, Beiyan; Korzh, Vladimir; Lodish, Harvey F; Lim, Bing

2009-04-01

The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response. To ensure a robust and precise response to cellular signals, p53 gene expression must be tightly regulated from the transcriptional to the post-translational levels. Computational predictions suggest that several microRNAs are involved in the post-transcriptional regulation of p53. Here we demonstrate that miR-125b, a brain-enriched microRNA, is a bona fide negative regulator of p53 in both zebrafish and humans. miR-125b-mediated down-regulation of p53 is strictly dependent on the binding of miR-125b to a microRNA response element in the 3' untranslated region of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. In contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebrafish brain. This phenotype can be rescued significantly by either an ablation of endogenous p53 function or ectopic expression of miR-125b in zebrafish. Interestingly, miR-125b is down-regulated when zebrafish embryos are treated with gamma-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses the increase of p53 and stress-induced apoptosis. Together, our study demonstrates that miR-125b is an important negative regulator of p53 and p53-induced apoptosis during development and during the stress response.

MicroRNA-125b is a novel negative regulator of p53

PubMed Central

Le, Minh T.N.; Teh, Cathleen; Shyh-Chang, Ng; Xie, Huangming; Zhou, Beiyan; Korzh, Vladimir; Lodish, Harvey F.; Lim, Bing

2009-01-01

The p53 transcription factor is a key tumor suppressor and a central regulator of the stress response. To ensure a robust and precise response to cellular signals, p53 gene expression must be tightly regulated from the transcriptional to the post-translational levels. Computational predictions suggest that several microRNAs are involved in the post-transcriptional regulation of p53. Here we demonstrate that miR-125b, a brain-enriched microRNA, is a bona fide negative regulator of p53 in both zebrafish and humans. miR-125b-mediated down-regulation of p53 is strictly dependent on the binding of miR-125b to a microRNA response element in the 3′ untranslated region of p53 mRNA. Overexpression of miR-125b represses the endogenous level of p53 protein and suppresses apoptosis in human neuroblastoma cells and human lung fibroblast cells. In contrast, knockdown of miR-125b elevates the level of p53 protein and induces apoptosis in human lung fibroblasts and in the zebrafish brain. This phenotype can be rescued significantly by either an ablation of endogenous p53 function or ectopic expression of miR-125b in zebrafish. Interestingly, miR-125b is down-regulated when zebrafish embryos are treated with γ-irradiation or camptothecin, corresponding to the rapid increase in p53 protein in response to DNA damage. Ectopic expression of miR-125b suppresses the increase of p53 and stress-induced apoptosis. Together, our study demonstrates that miR-125b is an important negative regulator of p53 and p53-induced apoptosis during development and during the stress response. PMID:19293287

Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis.

PubMed

Meresman, G F; Vighi, S; Buquet, R A; Contreras-Ortiz, O; Tesone, M; Rumi, L S

2000-10-01

To evaluate and compare spontaneous apoptosis and Bcl-2 and Bax expression in eutopic endometrium from women with and without endometriosis. Apoptosis and Bcl-2 and Bax expression were examined in eutopic endometrium from women with and without endometriosis. Instituto de Biología y Medicina Experimental-CONICET, Department of Gynecology and Department of Gynecological Pathology, Clínicas University Hospital, Buenos Aires, Argentina. Women with untreated endometriosis (n = 14) and controls (n = 16). Collection of endometrial samples during diagnostic or therapeutic laparoscopy. Apoptotic cells were detected with use of the dUTP nick-end labeling (TUNEL) assay; Bcl-2 and Bax expressions were assessed with use of immunohistochemical techniques. Spontaneous apoptosis was significantly lower in eutopic endometrium from patients with endometriosis, compared with healthy controls (2.26 +/- 0.53 and 9.37 +/- 1.69 apoptotic cells/field, respectively) and was independent of cycle phase. An increased expression of Bcl-2 protein was found in proliferative eutopic endometrium from patients with endometriosis. Bax expression was absent in proliferative endometrium, whereas there was an increase in its expression in secretory endometrium from both patients and controls. Women with endometriosis show decreased number of apoptotic cells in eutopic endometrium. The abnormal survival of endometrial cells may result in their continuing growth into ectopic locations.

Ectopic expression of Msx-2 in posterior limb bud mesoderm impairs limb morphogenesis while inducing BMP-4 expression, inhibiting cell proliferation, and promoting apoptosis.

PubMed

Ferrari, D; Lichtler, A C; Pan, Z Z; Dealy, C N; Upholt, W B; Kosher, R A

1998-05-01

During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell proliferation. Thus, a balance between cell proliferation and programmed cell death may play a very important role in maintaining the activity of the AER. Copyright 1998 Academic Press.

miR-320a regulates cell proliferation and apoptosis in multiple myeloma by targeting pre-B-cell leukemia transcription factor 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Yinghao; Department of Hematology, Affiliated Hospital of Guizhou Medical University, The Hematopoietic Stem Cell Transplant Center of Guizhou Province, Blood Diseases Diagnosis and Treatment Center of Guizhou Province, Guiyang, 550004, Guizhou Province; Wu, Depei, E-mail: wudepei@medmail.com.cn

2016-05-13

Aberrant expression of microRNAs (miRNAs) is implicated in cancer development and progression. While miR-320a is reported to be deregulated in many malignancy types, its biological role in multiple myeloma (MM) remains unclear. Here, we observed reduced expression of miR-320a in MM samples and cell lines. Ectopic expression of miR-320a dramatically suppressed cell viability and clonogenicity and induced apoptosis in vitro. Mechanistic investigation led to the identification of Pre-B-cellleukemia transcription factor 3 (PBX3) as a novel and direct downstream target of miR-320a. Interestingly, reintroduction of PBX3 abrogated miR-320a-induced MM cell growth inhibition and apoptosis. In a mouse xenograft model, miR-320a overexpression inhibitedmore » tumorigenicity and promoted apoptosis. Our findings collectively indicate that miR-320a inhibits cell proliferation and induces apoptosis in MM cells by directly targeting PBX3, supporting its utility as a novel and potential therapeutic agent for miRNA-based MM therapy. -- Highlights: •Expression of miR-320a in MM cell induces apoptosis in vitro. •miR-320a represses PBX3 via targeting specific sequences in the 3′UTR region. •Exogenous expression of PBX3 reverses the effects of miR-320a in inhibiting MM cell growth and promoting apoptosis. •Overexpression of miR-320a inhibits tumor growth and increases apoptosis in vivo.« less

Ectopic expression of necdin induces differentiation of mouse neuroblastoma cells.

PubMed

Kobayashi, Masakatsu; Taniura, Hideo; Yoshikawa, Kazuaki

2002-11-01

Necdin is expressed predominantly in postmitotic neurons, and ectopic expression of this protein strongly suppresses cell growth. Necdin has been implicated in the pathogenesis of Prader-Willi syndrome, a human neurodevelopmental disorder associated with genomic imprinting. Here we demonstrate that ectopic expression of necdin induces a neuronal phenotype in neuroblastoma cells. Necdin was undetectable in mouse neuroblastoma N1E-115 cells under undifferentiated and differentiated conditions. N1E-115 cells transfected with necdin cDNA showed morphological differentiation such as neurite outgrowth and expression of the synaptic marker proteins synaptotagmin and synaptophysin. In addition, Western blot analysis of the retinoblastoma protein (Rb) family members Rb, p130, and p107 revealed that necdin cDNA transfectants contained an increased level of p130 and a reduced level of p107, a pattern seen in differentiated G(0) cells. The transcription factors E2F1 and E2F4 physically interacted with necdin via their carboxyl-terminal transactivation domains, but only E2F1 abrogated necdin-induced growth arrest and neurite outgrowth of neuroblastoma cells. Overexpression of E2F1 in differentiated N1E-115 cells induced apoptosis, which was antagonized by co-expression of necdin. These results suggest that necdin promotes the differentiation and survival of neurons through its antagonistic interactions with E2F1.

The Nuclear Orphan Receptor NR4A1 is Involved in the Apoptotic Pathway Induced by LPS and Simvastatin in RAW 264.7 Macrophages.

PubMed

Kim, Yong Chan; Song, Seok Bean; Lee, Sang Kyu; Park, Sang Min; Kim, Young Sang

2014-04-01

Macrophage death plays a role in several physiological and inflammatory pathologies such as sepsis and arthritis. In our previous work, we showed that simvastatin triggers cell death in LPS-activated RAW 264.7 mouse macrophage cells through both caspase-dependent and independent apoptotic pathways. Here, we show that the nuclear orphan receptor NR4A1 is involved in a caspase-independent apoptotic process induced by LPS and simvastatin. Simvastatin-induced NR4A1 expression in RAW 264.7 macrophages and ectopic expression of a dominant-negative mutant form of NR4A1 effectively suppressed both DNA fragmentation and the disruption of mitochondrial membrane potential (MMP) during LPS- and simvastatin-induced apoptosis. Furthermore, apoptosis was accompanied by Bcl-2-associated X protein (Bax) translocation to the mitochondria. Our findings suggest that NR4A1 expression and mitochondrial translocation of Bax are related to simvastatin-induced apoptosis in LPS-activated RAW 264.7 macrophages.

Arctigenin enhances chemosensitivity to cisplatin in human nonsmall lung cancer H460 cells through downregulation of survivin expression.

PubMed

Wang, Huan-qin; Jin, Jian-jun; Wang, Jing

2014-01-01

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin-mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin-treated non-small-cell lung cancer (NSCLC) H460 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin-V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose-dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase-3 and poly(ADP-ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin-induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell-cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina-tion with chemotherapeutic agents for NSLC. © 2013 Wiley Periodicals, Inc.

NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin.

PubMed

Seo, Seung Un; Kim, Tae Hwan; Kim, Dong Eun; Min, Kyoung-Jin; Kwon, Taeg Kyu

2017-10-01

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Drosophila MOF regulates DIAP1 and induces apoptosis in a JNK dependent pathway.

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Koteswara Rao, G; Bag, Indira; Bhadra, Utpal; Pal-Bhadra, Manika

2016-03-01

Histone modulations have been implicated in various cellular and developmental processes where in Drosophila Mof is involved in acetylation of H4K16. Reduction in the size of larval imaginal discs is observed in the null mutants of mof with increased apoptosis. Deficiency involving Hid, Reaper and Grim [H99] alleviated mof (RNAi) induced apoptosis in the eye discs. mof (RNAi) induced apoptosis leads to activation of caspases which is suppressed by over expression of caspase inhibitors like P35 and Diap1 clearly depicting the role of caspases in programmed cell death. Also apoptosis induced by knockdown of mof is rescued by JNK mutants of bsk and tak1 indicating the role of JNK in mof (RNAi) induced apoptosis. The adult eye ablation phenotype produced by ectopic expression of Hid, Rpr and Grim, was restored by over expression of Mof. Accumulation of Mof at the Diap1 promoter 800 bp upstream of the transcription start site in wild type larvae is significantly higher (up to twofolds) compared to mof (1) mutants. This enrichment coincides with modification of histone H4K16Ac indicating an induction of direct transcriptional up regulation of Diap1 by Mof. Based on these results we propose that apoptosis triggered by mof (RNAi) proceeds through a caspase-dependent and JNK mediated pathway.

ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Komaragiri, Shravan Kumar; Bostanthirige, Dhanushka H.; Morton, Derrick J.

Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, wemore » examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. - Highlights: • ID4 expression induces AR expression in PC3 cells, which generally lack AR. • ID4 expression increased apoptosis and decreased cell proliferation and invasion. • Overexpression of ID4 reduces tumor growth of subcutaneous xenografts in vivo. • ID4 induces p21 and FKBP51 expression- co-factors of AR tumor suppressor activity.« less

The Bladder Tumor Suppressor Protein TERE1 (UBIAD1)Modulates Cell Cholesterol: Implications for Tumor Progression

PubMed Central

McGarvey, Terry; Wang, Huiyi; Lal, Priti; Puthiyaveettil, Raghunath; Tomaszewski, John; Sepulveda, Jorge; Labelle, Ed; Weiss, Jayne S.; Nickerson, Michael L.; Kruth, Howard S.; Brandt, Wolfgang; Wessjohann, Ludger A.; Malkowicz, S. Bruce

2011-01-01

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression. PMID:21740188

Noxa/Mcl-1 Balance Regulates Susceptibility of Cells to Camptothecin-Induced Apoptosis1

PubMed Central

Mei, Yide; Xie, Chongwei; Xie, Wei; Tian, Xu; Li, Mei; Wu, Mian

2007-01-01

Although camptothecin (CPT) has been reported to induce apoptosis in various cancer cells, the molecular details of this regulation remain largely unknown. In this study, we demonstrate that BH3-only protein Noxa is upregulated during CPT-induced apoptosis, which is independent of p53. In addition, we show that phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is responsible for Noxa's induction. Luciferase assay and cAMP response element binding protein (CREB) knockdown experiments further demonstrate that CREB is involved in the transcriptional upregulation of Noxa. Moreover, blocking Noxa expression using specific small interfering ribonucleic acid (siRNA) significantly reduces the apoptosis in response to CPT, indicating that Noxa is an essential mediator for CPT-induced apoptosis. Interestingly, antiapoptotic Mcl-1 was also upregulated through PI3K/Akt signaling pathway upon CPT treatment. Using immunoprecipitation assay, Noxa was found to interact with Mcl-1 in the presence or absence of CPT. Knockdown of Mcl-1 expression by short hairpin ribonucleic acid (shRNA) was shown to potentiate CPT-induced apoptosis. Consistently, ectopic overexpression of Mcl-1 rescued cells from apoptosis induced by CPT. Cells coexpressing Noxa and Mcl-1 at different ratio correlates well with the extent of apoptosis, suggesting that the balance between Noxa and Mcl-1 may determine the susceptibility of HeLa cells to CPT-induced apoptosis. PMID:17971907

Tunicamycin promotes apoptosis in leukemia cells through ROS generation and downregulation of survivin expression.

PubMed

Lim, Eun Jin; Heo, Jeonghoon; Kim, Young-Ho

2015-08-01

Tunicamycin (TN), one of the endoplasmic reticulum stress inducers, has been reported to inhibit tumor cell growth and exhibit anticarcinogenic activity. However, the mechanism by which TN initiates apoptosis remains poorly understood. In the present study, we investigated the effect of TN on the apoptotic pathway in U937 cells. We show that TN induces apoptosis in association with caspase-3 activation, generation of reactive oxygen species (ROS), and downregulation of survivin expression. P38 MAPK (mitogen-activated protein kinase) and the generation of ROS signaling pathway play crucial roles in TN-induced apoptosis in U937 cells. We hypothesized that TN-induced activation of p38 MAPK signaling pathway is responsible for cell death. To test this hypothesis, we selectively inhibited MAPK during treatment with TN. Our data demonstrated that inhibitor of p38 (SB), but not ERK (PD) or JNK (SP), partially maintained apoptosis during treatment with TN. Pre-treatment with NAC and GSH markedly prevented cell death, suggesting a role for ROS in this process. Ectopic expression of survivin in U937 cells attenuated TN-induced apoptosis by suppression of caspase-3 cleavage, mitochondrial membrane potential, and cytochrome c release in U937 cells. Taken together, our results show that TN modulates multiple components of the apoptotic response of human leukemia cells and raise the possibility of a novel therapeutic strategy for hematological malignancies.

Activated Hippo/Yes-Associated Protein Pathway Promotes Cell Proliferation and Anti-apoptosis in Endometrial Stromal Cells of Endometriosis.

PubMed

Song, Yong; Fu, Jing; Zhou, Min; Xiao, Li; Feng, Xue; Chen, Hengxi; Huang, Wei

2016-04-01

The imbalance in cell proliferation and apoptosis is considered an important role in the pathogenesis of endometriosis, but the exact mechanisms remains unclear. A newly established signaling pathway–Hippo/Yes-associated protein (YAP) pathway plays a critical role in the proliferation and apoptosis processes. However, studies focusing on Hippo/YAP pathway and endometriosis are lacking. The objective was to explore the function of the Hippo/YAP pathway in endometriosis. The expression of YAP was first investigated in endometrium of women with or without endometriosis. The role of YAP in cell proliferation and apoptosis is identified by transfection of endometrial stromal cells (ESCs) in vitro, subsequent Verteporfin treatments in eutopic ESCs in vitro, and endometriosis animal model of nude mice in vivo. Our results revealed that increased expression of YAP and decreased expression of p-YAP in ectopic and eutopic endometrium compared with normal endometrium. YAP knockdown in eutopic ESCs decreased cell proliferation and enhanced cell apoptosis companied with decreased expression of TEAD1, CTGF, and B-cell lymphoma/leukemia (BCL)-2; whereas overexpression of YAP resulted in increased proliferation and decreased apoptosis of normal ESCs with increased expression of TEAD1, CTGF, and BCL-2. By chromatin immunoprecipitation qPCR CTGF and BCL-2 were identified as directly downstream target genes of YAP-TEAD1 active complex. Eutopic ESCs treated with Verteporfin revealed decreased proliferation and enhanced apoptosis whereas in endometriosis animal models of nude mice treated with Verteporfin, the size of endometriotic lesions was significantly reduced. Our study suggests that the Hippo/YAP-signaling pathway plays a critical role in the pathogenesis of endometriosis and should present a novel therapeutic method against endometriosis.

The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition

PubMed Central

Li, Bo; Ren, Hui; Yue, Ping; Chen, Mingwei; Khuri, Fadlo R.; Sun, Shi-Yong

2012-01-01

API-1 is a novel small molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation, and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of c-FLIP levels and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of DR4 or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis, but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1, but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Since other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. PMID:22345097

Targeting Bcl-2 stability to sensitize cells harboring oncogenic ras.

PubMed

Peng, Bo; Ganapathy, Suthakar; Shen, Ling; Huang, Junchi; Yi, Bo; Zhou, Xiaodong; Dai, Wei; Chen, Changyan

2015-09-08

The pro-survival factor Bcl-2 and its family members are critical determinants of the threshold of the susceptibility of cells to apoptosis. Studies are shown that cells harboring an oncogenic ras were extremely sensitive to the inhibition of protein kinase C (PKC) and Bcl-2 could antagonize this apoptotic process. However, it remains unrevealed how Bcl-2 is being regulated in this apoptotic process. In this study, we investigate the role of Bcl-2 stability in sensitizing the cells harboring oncogenic K-ras to apoptosis triggered by PKC inhibitor GO6976. We demonstrated that Bcl-2 in Swiss3T3 cells ectopically expressing or murine lung cancer LKR cells harboring K-ras rapidly underwent ubiquitin-dependent proteasome pathway after the treatment of GO6976, accompanied with induction of apoptosis. In this process, Bcl-2 formed the complex with Keap-1 and Cul3. The mutation of serine-17 and deletion of BH-2 or 4 was required for Bcl-2 ubiquitination and degradation, which elevate the signal threshold for the induction of apoptosis in the cells following PKC inhibition. Thus, Bcl-2 appears an attractive target for the induction of apoptosis by PKC inhibition in cancer cells expressing oncogenic K-ras.

Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

PubMed

Wang, Qianli; Lingel, Amy; Geiser, Vicki; Kwapnoski, Zachary; Zhang, Luwen

2017-10-15

Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers. IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA damage-mediated cellular stresses. This seems to be the first report that p53 activates a viral oncogene; therefore, the discovery would be interesting to a broad readership from the fields of oncology to virology. Copyright © 2017 American Society for Microbiology.

miR-340 alleviates chemoresistance of osteosarcoma cells by targeting ZEB1.

PubMed

Yan, Haibin; Zhang, Bingyun; Fang, Chongbin; Chen, Liqiu

2018-06-01

Chemoresistance during treatment of osteosarcoma (OS) is attracting more and more attention as the main clinical obstacle. The purpose of this study was to elucidate the role of miR-340 in chemoresistance of OS. Plasmid construction and transfection, miRNA arrays, PCR analyses, and western blot analysis, as well as MTT, apoptosis, and luciferase assays were carried out in MG-63 cells and MG-63/cisplatin (DDP)-resistant cells. The results showed that miR-340 was downregulated in OS tissues and drug-resistant OS cells. Moreover, a negative correlation was observed between miR-340 and ZEB1 expression in OS tissues. Forced expression of miR-340 in drug-resistant OS cells significantly reduced multidrug resistance-1 and P-gp expression. Overexpression of miR-340 enhanced sensitivity to DDP by inhibiting viability and promoting apoptosis. The luciferase assay and western blot analysis identified ZEB1 as a direct target of miR-340, and miR-340 negatively regulated ZEB1 expression. Ectopic expression of ZEB1 reversed the effects of miR-340 on P-gp expression, cell viability, and apoptosis. miR-340 alleviated chemoresistance of OS cells by targeting ZEB1. Our results indicate that targeting miR-340 may be a potential therapeutic approach to treat drug-resistant OS.

Mechanism of Telomerase Activation by v-Rel and Its Contribution to Transformation

PubMed Central

Hrdličková, Radmila; Nehyba, Jiří; Liss, Andrew S.; Bose, Henry R.

2006-01-01

Telomerase is activated during the transformation of lymphoid cells and fibroblasts by v-Rel, the oncogenic member of the Rel/NF-κB family of transcription factors. v-Rel-transformed cell lines have longer telomeres than untransformed chicken lymphoid cells and have high levels of telomerase activity. v-Rel-mediated activation of telomerase is achieved by multiple mechanisms. The expression of the gene encoding the catalytic subunit of telomerase (TERT) was directly upregulated by v-Rel. Moreover, the expression of v-Rel altered the ratio of alternatively spliced and full-length TERT transcripts in favor of the full-length forms. The activation of telomerase by v-Rel in lymphocytes was also accompanied by inactivation of nuclear inhibitors. The inhibition of telomerase activity in v-Rel-transformed cell lines led to apoptosis within 24 h. The expression of v-Rel in a macrophage cell line resulted in elevated levels of reactive oxygen species (ROS), increased telomerase activity, and increased sensitivity to telomerase inhibitors. In contrast, the ectopic expression of TERT decreased the extent of apoptosis induced by ROS. The activation of telomerase by v-Rel may, therefore, partially protect the transformed cells from apoptosis induced by ROS. PMID:16352553

Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras.

PubMed

Fenton, R G; Hixon, J A; Wright, P W; Brooks, A D; Sayers, T J

1998-08-01

The ras oncogene plays an important role in the multistep progression to cancer by activation of signal transduction pathways that contribute to aberrant growth regulation. Although many of these effects are cell autonomous, the ras oncogene also regulates the expression of genes that alter host/tumor interactions. We now extend the mechanisms through which ras promotes tumor survival by demonstrating that oncogenic Ras inhibits expression of the fas gene and renders Ras-transformed cells resistant to Fas-induced apoptosis. A panel of Ras-transformed clones exhibited a marked inhibition in fas mRNA and Fas cell surface expression as compared with untransformed parental cell lines. Fas expression was induced by culture in the presence of IFN-gamma + tumor necrosis factor alpha; however, the maximal level attained in Ras transformants was approximately 10-fold below the level of untransformed cells. Whereas untransformed cells were sensitive to apoptotic death induced by cross-linking surface Fas (especially after cytokine treatment), Ras-transformed cells were very resistant to Fas-induced death even under the most stringent assay conditions. To demonstrate that this resistance was mediated by oncogenic Ras and not secondary genetic events, pools of Ras-transformed cells were generated using a highly efficient retroviral transduction technique. Transformed pools were assayed 6 days after infection and demonstrated a marked decrease in fas gene expression and Fas-mediated apoptosis. Oncogenic Ras did not promote general resistance to apoptosis, because ectopic expression of a fas cDNA in Ras-transformed cells restored sensitivity to Fas-induced apoptosis. These data indicate that oncogenic Ras inhibits basal levels of expression of the fas gene, and although cytokine signal transduction pathways are functional in these cells, the level of surface Fas expression remains below the threshold required for induction of apoptosis. These data identify a mechanism by which Ras-transformed cells may escape from host-mediated immune destruction.

TNFRp55 deficiency promotes the development of ectopic endometriotic-like lesions in mice.

PubMed

Vallcaneras, Sandra; Ghersa, Federica; Bastón, Juan; Delsouc, María Belén; Meresman, Gabriela; Casais, Marilina

2017-09-01

Endometriosis is an inflammatory disease depending on estradiol, with TNF-α being one of the most representative cytokines involved in its pathogenesis. TNF-α acts through its bond to the TNFRp55 and TNFRp75 membrane receptors. The aim of this study was to analyze the effect of the TNFRp55 deficiency on the development of ectopic endometriotic-like lesions. Endometriosis was induced surgically in mice of the C57BL/6 strain, wild type (WT) and TNFRp55-/- (KO). After four weeks, the peritoneal fluid was collected and the lesions were counted, measured with a caliper, removed, weighed, fixed or kept at -80°C. We evaluated the cell proliferation by proliferating cell nuclear antigen (PCNA) immunohistochemistry and apoptosis by TUNEL technique in the ectopic lesions. MMP-2 and MMP-9 activities (factors involved in invasiveness) were measured by zymography in the peritoneal fluid; estradiol and progesterone levels were measured by radioimmunoassay in the lesions and in the peritoneal fluid. We found that in KO animals the mean number of lesions established per mouse, the lesion volume, weight and cell proliferation increased and apoptosis decreased. In addition, the activity of MMP-2 and the estradiol level increased, whereas the progesterone level was not significantly modified. In conclusion, the deficiency of TNFRp55 promoted the establishment and development of endometriosis through an increase in the lesion size and high levels of estradiol which correlate with an increase in the MMP-2 activity. This is evidence of the possible association of the deregulation of the TNFRp55 expression and the survival of the endometriotic tissue in ectopic sites. © 2017 Society for Endocrinology.

Chemotherapy resistance and metastasis-promoting effects of thyroid hormone in hepatocarcinoma cells are mediated by suppression of FoxO1 and Bim pathway

PubMed Central

Chi, Hsiang-Cheng; Chen, Shen-Liang; Cheng, Yi-Hung; Lin, Tzu-Kang; Tsai, Chung-Ying; Tsai, Ming-Ming; Lin, Yang-Hsiang; Huang, Ya-Hui; Lin, Kwang-Huei

2016-01-01

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and systemic chemotherapy is the major treatment strategy for late-stage HCC patients. Poor prognosis following chemotherapy is the general outcome owing to recurrent resistance. Recent studies have suggested that in addition to cytotoxic effects on tumor cells, chemotherapy can induce an alternative cascade that supports tumor growth and metastasis. In the present investigation, we showed that thyroid hormone (TH), a potent hormone-mediating cellular differentiation and metabolism, acts as an antiapoptosis factor upon challenge of thyroid hormone receptor (TR)-expressing HCC cells with cancer therapy drugs, including cisplatin, doxorubicin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TH/TR signaling promoted chemotherapy resistance through negatively regulating the pro-apoptotic protein, Bim, resulting in doxorubicin-induced metastasis of chemotherapy-resistant HCC cells. Ectopic expression of Bim in hepatoma cells challenged with chemotherapeutic drugs abolished TH/TR-triggered apoptosis resistance and metastasis. Furthermore, Bim expression was directly transactivated by Forkhead box protein O1 (FoxO1), which was negatively regulated by TH/TR. TH/TR suppressed FoxO1 activity through both transcriptional downregulation and nuclear exclusion of FoxO1 triggered by Akt-mediated phosphorylation. Ectopic expression of the constitutively active FoxO1 mutant, FoxO1-AAA, but not FoxO1-wt, diminished the suppressive effect of TH/TR on Bim. Our findings collectively suggest that expression of Bim is mediated by FoxO1 and indirectly downregulated by TH/TR, leading to chemotherapy resistance and doxorubicin-promoted metastasis of hepatoma cells. PMID:27490929

Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins.

PubMed

Chowdhury, Indrajit; Thompson, Winston E; Welch, Crystal; Thomas, Kelwyn; Matthews, Roland

2013-12-01

Mammalian ovarian follicular development is tightly regulated by crosstalk between cell death and survival signals, which include both endocrine and intra-ovarian regulators. Whether the follicle ultimately ovulates or undergoes atresia is dependent on the expression and actions of factors promoting follicular cell proliferation, differentiation or apoptosis. Prohibitin (PHB) is a highly conserved, ubiquitous protein that is abundantly expressed in granulosa cells (GCs) and associated with GC differentiation and apoptosis. The current study was designed to characterize the regulation of anti-apoptotic and pro-apoptotic factors in undifferentiated rat GCs (gonadotropin independent phase) governed by PHB. Microarray technology was initially employed to identify potential apoptosis-related genes, whose expression levels within GCs were altered by either staurosporine (STS) alone or STS in presence of ectopically over-expressed PHB. Next, immunoblot studies were performed to examine the expression patterns of selective Bcl-2 family members identified by the microarray analysis, which are commonly regulated in the intrinsic-apoptotic pathway. These studies were designed to measure protein levels of Bcl2 family in relation to expression of the acidic isoform (phosphorylated) PHB and the components of MEK-Erk1/2 pathway. These studies indicated that over-expression of PHB in undifferentiated GCs inhibit apoptosis which concomitantly results in an increased level of the anti-apoptotic proteins Bcl2 and Bclxl, reduced release of cytochrome c from mitochondria and inhibition of caspase-3 activity. In contrast, silencing of PHB expression resulted in change of mitochondrial morphology from the regular reticular network to a fragmented form, which enhanced sensitization of these GCs to the induction of apoptosis. Collectively, these studies have provided new insights on the PHB-mediated anti-apoptotic mechanism, which occurs in undifferentiated GCs through a PHB → Mek-Erk1/2 → Bcl/Bcl-xL pathway and may have important clinical implications.

Obatoclax analog SC-2001 inhibits STAT3 phosphorylation through enhancing SHP-1 expression and induces apoptosis in human breast cancer cells.

PubMed

Liu, Chun-Yu; Su, Jung-Chen; Ni, Mei-Huei; Tseng, Ling-Ming; Chu, Pei-Yi; Wang, Duen-Shian; Tai, Wei-Tien; Kao, Yuan-Ping; Hung, Man-Hsin; Shiau, Chung-Wai; Chen, Kuen-Feng

2014-07-01

Interfering oncogenic STAT3 signaling is a promising anti-cancer strategy. We examined the efficacy and drug mechanism of an obatoclax analog SC-2001, a novel STAT3 inhibitor, in human breast cancer cells. Human breast cancer cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and western blot. Signaling pathways were assessed by western blot. In vivo efficacy of SC-2001 was tested in xenograft nude mice. SC-2001 inhibited cell growth and induced apoptosis in association with downregulation of p-STAT3 (Tyr 705) in breast cancer cells. STAT3-regulated proteins, including Mcl-1, survivin, and cyclin D1, were repressed by SC-2001. Over-expression of STAT3 in MDA-MB-468 cells protected cells from SC-2001-induced apoptosis. Moreover, SC-2001 enhanced the expression of protein tyrosine phosphatase SHP-1, a negative regulator of STAT3. Furthermore, the enhanced SHP-1 expression, in conjunction with increased SHP-1 phosphatase activity, was mediated by upregulated transcription by RFX-1. Chromatin immunoprecipitation assay revealed that SC-2001 increased the binding capacity of RFX-1 to the SHP-1 promoter. Knockdown of either RFX-1 or SHP-1 reduced SC-2001-induced apoptosis, whereas ectopic expression of RFX-1 increased SHP-1 expression and enhanced the apoptotic effect of SC-2001. Importantly, SC-2001 suppressed tumor growth in association with enhanced RFX-1 and SHP-1 expression and p-STAT3 downregulation in MDA-MB-468 xenograft tumors. SC-2001 induced apoptosis in breast cancer cells, an effect that was mediated by RFX-1 upregulated SHP-1 expression and SHP-1-dependent STAT3 inactivation. Our study indicates targeting STAT3 signaling pathway may be a useful approach for the development of targeted agents for anti-breast cancer.

Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation.

PubMed

Woo, Seon Min; Min, Kyoung-Jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Chun, Kyung-Soo; Kim, Young Ho; Lee, Tae-Jin; Kim, Sang Hyun; Choi, Yung Hyun; Chang, Jong-Soo; Kwon, Taeg Kyu

2014-03-25

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

PubMed

Danthi, Pranav; Coffey, Caroline M; Parker, John S L; Abel, Ty W; Dermody, Terence S

2008-12-01

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

Human Chorionic Gonadotropin and Breast Cancer

PubMed Central

Schüler-Toprak, Susanne; Treeck, Oliver; Ortmann, Olaf

2017-01-01

Breast cancer is well known as a malignancy being strongly influenced by female steroids. Pregnancy is a protective factor against breast cancer. Human chorionic gonadotropin (HCG) is a candidate hormone which could mediate this antitumoral effect of pregnancy. For this review article, all original research articles on the role of HCG in breast cancer were considered, which are listed in PubMed database and were written in English. The role of HCG in breast cancer seems to be a paradox. Placental heterodimeric HCG acts as a protective agent by imprinting a permanent genomic signature of the mammary gland determining a refractory condition to malignant transformation which is characterized by cellular differentiation, apoptosis and growth inhibition. On the other hand, ectopic expression of β-HCG in various cancer entities is associated with poor prognosis due to its tumor-promoting function. Placental HCG and ectopically expressed β-HCG exert opposite effects on breast tumorigenesis. Therefore, mimicking pregnancy by treatment with HCG is suggested as a strategy for breast cancer prevention, whereas targeting β-HCG expressing tumor cells seems to be an option for breast cancer therapy. PMID:28754015

The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition.

PubMed

Li, Bo; Ren, Hui; Yue, Ping; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2012-04-01

API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. 2012 AACR

miR-24-3p Suppresses Malignant Behavior of Lacrimal Adenoid Cystic Carcinoma by Targeting PRKCH to Regulate p53/p21 Pathway.

PubMed

Zhang, Ming-Xue; Zhang, Jie; Zhang, Hong; Tang, Hua

2016-01-01

MicroRNA (miRNA) may function as an oncogene or a tumor suppressor in tumorigenesis. However, the mechanism of miRNAs in adenoid cystic carcinoma (ACC) is unclear. Here, we provide evidence that miR-24-3p was downreglated and functions as a tumor suppressor in human lacrimal adenoid cystic carcinoma by suppressing proliferation and migration/invasion while promoting apoptosis. miR-24-3p down-regulated protein kinase C eta (PRKCH) by binding to its untranslated region (3'UTR). PRKCH increased the of the cell growth and migration/invasion in ACC cells and suppressed the expression of p53 and p21 in both mRNA and protein level. The overexpression of miR-24-3p decreased its malignant phenotype. Ectopic expression of PRKCH counteracted the suppression of malignancy induced by miR-24-3p, as well as ectopic expression of miR-24-3p rescued the suppression of PRKCH in the p53/p21 pathway. These results suggest that miR-24-3p promotes the p53/p21 pathway by down-regulating PRKCH expression in lacrimal adenoid cystic carcinoma cells.

Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle.

PubMed

Lyons, John D; Klingensmith, Nathan J; Otani, Shunsuke; Mittal, Rohit; Liang, Zhe; Ford, Mandy L; Coopersmith, Craig M

2017-12-01

Cell production and death are tightly regulated in the rapidly renewing gut epithelium, with proliferation confined to crypts and apoptosis occurring in villi and crypts. This study sought to determine how stress alters these compartmentalized processes. Wild-type mice made septic via cecal ligation and puncture had decreased crypt proliferation and increased crypt and villus apoptosis. Fabpi -TAg mice expressing large T-antigen solely in villi had ectopic enterocyte proliferation with increased villus apoptosis in unmanipulated animals. Septic fabpi -TAg mice had an unexpected increase in villus proliferation compared with unmanipulated littermates, whereas crypt proliferation was decreased. Cell cycle regulators cyclin D1 and cyclin D2 were decreased in jejunal tissue in septic transgenic mice. In contrast, villus and crypt apoptosis were increased in septic fabpi -TAg mice. To examine the relationship between apoptosis and proliferation in a compartment-specific manner, fabpi -TAg mice were crossed with fabpl -Bcl-2 mice, resulting in expression of both genes in the villus but Bcl-2 alone in the crypt. Septic bi-transgenic animals had decreased crypt apoptosis but had a paradoxical increase in villus apoptosis compared with septic fabpi -TAg mice, associated with decreased proliferation in both compartments. Thus, sepsis unmasks compartment-specific proliferative and apoptotic regulation that is not present under homeostatic conditions.-Lyons, J. D., Klingensmith, N. J., Otani, S., Mittal, R., Liang, Z., Ford, M. L., Coopersmith, C. M. Sepsis reveals compartment-specific responses in intestinal proliferation and apoptosis in transgenic mice whose enterocytes re-enter the cell cycle. © FASEB.

Elevated expression of CD147 in patients with endometriosis and its role in regulating apoptosis and migration of human endometrial cells.

PubMed

Jin, Aihong; Chen, Hao; Wang, Chaoqun; Tsang, Lai Ling; Jiang, Xiaohua; Cai, Zhiming; Chan, Hsiao Chang; Zhou, Xiaping

2014-06-01

To examine the expression of CD147 in 60 human endometriosis lesions and how CD147 regulates migration and apoptosis in human uterine epithelial (HESs) cells. Experimental clinical study and laboratory-based investigation. Hospital and academic research center. Sixty women with chocolate cysts and 16 control women without endometriosis. Human uterine epithelial cells were treated with anti-CD147 antibody. Real-time polymerase chain reaction for detecting CD147 expression in 60 human endometriosis lesions; migration assay and CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) assay for cell functional investigation; Western blot for detecting protein levels; gelatin zymography for evaluating the activity of matrix metalloproteinase-2 (MMP-2) in cultured cells. Expression of CD147 was significantly higher in ectopic endometrial tissues from patients with endometriosis than in normal endometrial tissues. Interference with CD147 function led to decreased migration and cell viability in HESs cells. Surprisingly, MMP-2 expression and activity were not changed after treating HESs cells with anti-CD147 antibody. Further examination revealed that immunodepletion of CD147 induced apoptosis in HESs cells, leading to the activation of caspase 3 and poly(ADP-ribose) polymerase. The results of the present study suggest that abnormally high expression of CD147 in ovarian endometriosis lesions with enhanced cell survival (reduced apoptosis) and migration, in an MMP-2-independent manner, may underlie the progression of endometriosis in humans. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Role of MicroRNA-1 in Human Cancer and Its Therapeutic Potentials

PubMed Central

Han, Chao; Yu, Zujiang; Duan, Zhenfeng; Kan, Quancheng

2014-01-01

While the mechanisms of human cancer development are not fully understood, evidence of microRNA (miRNA, miR) dysregulation has been reported in many human diseases, including cancer. miRs are small noncoding RNA molecules that regulate posttranscriptional gene expression by binding to complementary sequences in the specific region of gene mRNAs, resulting in downregulation of gene expression. Not only are certain miRs consistently dysregulated across many cancers, but they also play critical roles in many aspects of cell growth, proliferation, metastasis, apoptosis, and drug resistance. Recent studies from our group and others revealed that miR-1 is frequently downregulated in various types of cancer. Through targeting multiple oncogenes and oncogenic pathways, miR-1 has been demonstrated to be a tumor suppressor gene that represses cancer cell proliferation and metastasis and promotes apoptosis by ectopic expression. In this review, we highlight recent findings on the aberrant expression and functional significance of miR-1 in human cancers and emphasize its significant values for therapeutic potentials. PMID:24949449

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis*

PubMed Central

Barnwal, Bhaskar; Karlberg, Helen; Mirazimi, Ali; Tan, Yee-Joo

2016-01-01

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells. PMID:26574543

Myostatin induces mitochondrial metabolic alteration and typical apoptosis in cancer cells

PubMed Central

Liu, Y; Cheng, H; Zhou, Y; Zhu, Y; Bian, R; Chen, Y; Li, C; Ma, Q; Zheng, Q; Zhang, Y; Jin, H; Wang, X; Chen, Q; Zhu, D

2013-01-01

Myostatin, a member of the transforming growth factor-β superfamily, regulates the glucose metabolism of muscle cells, while dysregulated myostatin activity is associated with a number of metabolic disorders, including muscle cachexia, obesity and type II diabetes. We observed that myostatin induced significant mitochondrial metabolic alterations and prolonged exposure of myostatin induced mitochondria-dependent apoptosis in cancer cells addicted to glycolysis. To address the underlying mechanism, we found that the protein levels of Hexokinase II (HKII) and voltage-dependent anion channel 1 (VDAC1), two key regulators of glucose metabolisms as well as metabolic stress-induced apoptosis, were negatively correlated. In particular, VDAC1 was dramatically upregulated in cells that are sensitive to myostatin treatment whereas HKII was downregulated and dissociated from mitochondria. Myostatin promoted the translocation of Bax from cytosol to mitochondria, and knockdown of VDAC1 inhibited myostatin-induced Bax translocation and apoptosis. These apoptotic changes can be partially rescued by repletion of ATP, or by ectopic expression of HKII, suggesting that perturbation of mitochondrial metabolism is causally linked with subsequent apoptosis. Our findings reveal novel function of myostatin in regulating mitochondrial metabolism and apoptosis in cancer cells. PMID:23412387

Inhibition of BET bromodomain-dependent XIAP and FLIP expression sensitizes KRAS-mutated NSCLC to pro-apoptotic agents

PubMed Central

Klingbeil, Olaf; Lesche, Ralf; Gelato, Kathy A; Haendler, Bernard; Lejeune, Pascale

2016-01-01

Non-small cell lung cancer (NSCLC) has the highest incidence of cancer-related death worldwide and a high medical need for more effective therapies. Small-molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1, I-BET762 and OTX-015 are active in a wide range of different cancer types, including lung cancer. Although their activity on oncogene expression such as c-Myc has been addressed in many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. Here we evaluated the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptosis response. Using a panel of 12 KRAS-mutated NSCLC models, we found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with downregulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. In the H1373 xenograft model, treatment with JQ1 significantly reduced tumor growth and downregulated the expression of c-Myc. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and we found the expression of the two key apoptosis regulators FLIP and XIAP to be highly BET dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Further we showed that combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether, these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment. They also demonstrate that BET inhibition primes NSCLC cells for induction of apoptosis and that a combination with pro-apoptotic compounds represents a valuable strategy to overcome treatment resistance. PMID:27607580

A long-range foresight for the medical application of apoptosis specifically induced by Dd-MRP4, Dictyostelium mitochondrial ribosomal protein S4, to cancer therapy.

PubMed

Maeda, Yasuo

2015-02-10

Apoptosis (programmed cell death) is regarded as ultimate differentiation of the cell. We have recently demonstrated that a targeted delivery of Dd-MRP4 (Dictyostelium mitochondrial ribosomal protein S4) suppresses specifically the proliferation of the human cancer cells, by inducing their apoptotic cell death (Chida et al., 2014, doi:10.1186/1475-2867-14-56). This amazing fact was discovered, simply based on the finding that Dd-MRP4 expression is absolutely required for transition of Dictyostelium cells from growth to differentiation (Chida et al., 2008, doi:10.1186/1471-2156-9-25; Maeda et al., 2013, doi:10.3390/biom3040943). Dd-MRP4 protein has quite unique structural characters, in that it is highly basic (pI: about 11.5) and interestingly has several nuclear-localization signals within the molecule. In this review, we introduce briefly the efficacy of several apoptosis-inducing substances reported thus far for cancer therapy, and speculate the possible mechanisms, by which apoptosis is specifically induced by Dd-MRP4, on the basis of its structural uniqueness. We also discuss several issues to be solved for the medical application of ectopically expressed Dd-MRP4 in human cancer cells.

ID4 promotes AR expression and blocks tumorigenicity of PC3 prostate cancer cells.

PubMed

Komaragiri, Shravan Kumar; Bostanthirige, Dhanushka H; Morton, Derrick J; Patel, Divya; Joshi, Jugal; Upadhyay, Sunil; Chaudhary, Jaideep

2016-09-09

Deregulation of tumor suppressor genes is associated with tumorigenesis and the development of cancer. In prostate cancer, ID4 is epigenetically silenced and acts as a tumor suppressor. In normal prostate epithelial cells, ID4 collaborates with androgen receptor (AR) and p53 to exert its tumor suppressor activity. Previous studies have shown that ID4 promotes tumor suppressive function of AR whereas loss of ID4 results in tumor promoter activity of AR. Previous study from our lab showed that ectopic ID4 expression in DU145 attenuates proliferation and promotes AR expression suggesting that ID4 dependent AR activity is tumor suppressive. In this study, we examined the effect of ectopic expression of ID4 on highly malignant prostate cancer cell, PC3. Here we show that stable overexpression of ID4 in PC3 cells leads to increased apoptosis and decreased cell proliferation and migration. In addition, in vivo studies showed a decrease in tumor size and volume of ID4 overexpressing PC3 cells, in nude mice. At the molecular level, these changes were associated with increased androgen receptor (AR), p21, and AR dependent FKBP51 expression. At the mechanistic level, ID4 may regulate the expression or function of AR through specific but yet unknown AR co-regulators that may determine the final outcome of AR function. Copyright © 2016 Elsevier Inc. All rights reserved.

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein.

PubMed

Yoneda-Kato, N; Fukuhara, S; Kato, J

1999-06-24

The NPM-MLF1 chimeric protein is produced by the t(3;5)(q25.1;q34) chromosomal translocation, which is associated with myelodysplastic syndrome (MDS) prior to progression into acute myeloid leukemia (AML). Here we report that K562 human leukemia cells ectopically expressing NPM-MLF1, but not those with wild-type MLF1, were gradually eliminated from the culture by undergoing apoptosis. NIH3T3 mouse fibroblasts engineered to overexpress NPM-MLF1 grew normally but serum deprivation triggered apoptotic cell death with slower kinetics than did other well-known apoptotic inducers such as c-Myc or E2F-1. Quantitative analysis of apoptotic induction confirmed that, neither NPM nor MLF1, but the NPM-MLF1 fusion protein was able to induce apoptosis. Analyses using a variety of deletion mutants of NPM-MLF1 revealed that induction of apoptosis required the N-terminal domain of MLF1 and the NPM domain containing nuclear localization signal and that removal of the NPM dimerization domain markedly impaired the ability to induce apoptosis. Co-expression of Bcl-2 rescued NIH3T3 fibroblasts from NPM-MLF1-mediated cell death without affecting the expression level or the subcellular localization of NPM-MLF1 and enabled cells to progress into S phase in low serum. These findings provide an NPM-MLF1-mediated novel mechanism of apoptotic induction and imply that NPM-MLFI in collaboration with anti-apoptotic oncoproteins may play an important role in multi-step progression from MDS to AML.

Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins

PubMed Central

Bernasconi, Michele; Remppis, Andrew; Fredericks, William J.; Rauscher, Frank J.; Schäfer, Beat W.

1996-01-01

The expression of a number of human paired box-containing (PAX) genes has been correlated with various types of tumors. Novel fusion genes encoding chimeric fusion proteins have been found in the pediatric malignant tumor alveolar rhabdomyosarcoma (RMS). They are generated by two chromosomal translocations t(2;13) and t(1;13) juxtaposing PAX3 or PAX7, respectively, with a forkhead domain gene FKHR. Here we describe that specific down-regulation of the t(2;13) translocation product in alveolar RMS cells by antisense oligonucleotides results in reduced cellular viability. Cells of embryonal RMS, the other major histiotype of this tumor, were found to express either wild type PAX3 or PAX7 at elevated levels when compared with primary human myoblasts. Treatment of corresponding embryonal RMS cells with antisense olignucleotides directed against the mRNA translational start site of either one of these two transcription factors similarly triggers cell death, which is most likely due to induction of apoptosis. Retroviral mediated ectopic expression of mouse Pax3 in a PAX7 expressing embryonal RMS cell line could partially rescue antisense induced apoptosis. These data suggest that the PAX3/FKHR fusion gene and wild-type PAX genes play a causative role in the formation of RMS and presumably other tumor types, possibly by suppressing the apoptotic program that would normally eliminate these cells. PMID:8917562

Decreased LRIG1 in Human Ovarian Cancer Cell SKOV3 Upregulates MRP-1 and Contributes to the Chemoresistance of VP16.

PubMed

Yang, Hua; Yao, Jun; Yin, Jiangpin; Wei, Xuan

2016-05-01

The leucine-rich repeats and immunoglobulin-like domains (LRIG) are used as tumor suppressors in clinical applications. Although the LRIG has been identified to manipulate the cell proliferation via various oncogenic receptor tyrosine kinases in diverse cancers, its role in multidrug resistance needs to be further elucidated, especially in human ovarian cancer. We herein established that the etoposide (VP16)-resistant SKOV3 human ovarian cancer cell clones (SKOV3/VP16 cells) and mRNA expression of LRIG1 were significantly reduced by the treatment of VP16 in a concentration-dependent manner. Moreover, downregulated LRIG1 in SKOV3 could enhance the colony formation and resist the inhibition of proliferation by VP16, leading to the elevated expression of Bcl-2 and decreased apoptosis of SKOV3. Interestingly, our results uncovered that the multidrug resistance-associated protein 1 (MRP-1) was upregulated for the chemoresistance of VP16. To overcome the chemoresistance of SKOV3, SKOV3/VP16 was ectopically expressed of LRIG1. We found that the inhibition of VP16 on colony formation and proliferation was remarkably enhanced with increased apoptosis in SKOV3/VP16. Furthermore, the expression of MRP-1 and Bcl-2 was also inhibited, suggesting that the LRIG1could negatively control MRP-1 and the apoptosis to improve the sensitivity of VP16-related chemotherapy.

Ectopic overexpression of LAPTM5 results in lysosomal targeting and induces Mcl-1 down-regulation, Bak activation, and mitochondria-dependent apoptosis in human HeLa cells

PubMed Central

Jun, Do Youn; Kim, Hyejin; Jang, Won Young; Lee, Ji Young; Fukui, Kiyoshi; Kim, Young Ho

2017-01-01

Human lysosomal-associated protein multispanning membrane 5 (LAPTM5) was identified by an ordered differential display-polymerase chain reaction (ODD-PCR) as an up-regulated cDNA fragment during 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced differentiation of U937 cells into monocytes/macrophages. After TPA-treatment, the levels of LAPTM5 mRNA and protein increased and reached a maximum at 18–36 h. In healthy human tissues, LAPTM5 mRNA was expressed at high levels in hematopoietic cells and tissues, at low levels in the lung and fetal liver, and was not detected in other non-hematopoietic tissues. LAPTM5 mRNA was detected in immature malignant cells of myeloid lineage, such as K562, HL-60, U937, and THP-1 cells, and in unstimulated peripheral T cells, but was absent or barely detectable in lymphoid malignant or non-hematopoietic malignant cells. The LAPTM5 level in HL-60 cells increased more significantly during TPA-induced monocyte/macrophage differentiation than during DMSO-induced granulocyte differentiation. Ectopic expression of GFP-LAPTM5 or LAPTM5 in HeLa cells exhibited the localization of LAPTM5 to the lysosome. In HeLa cells overexpressing LAPTM5, the Mcl-1 and Bid levels declined markedly and apoptosis was induced via Bak activation, Δψm loss, activation of caspase-9, -8 and -3, and PARP degradation without accompanying necrosis. However, these LAPTM5-induced apoptotic events except for the decline of Bid level were completely abrogated by concomitant overexpression of Mcl-1. The pan-caspase inhibitor (z-VAD-fmk) could suppress the LAPTM5-induced apoptotic sub-G1 peak by ~40% but failed to block the induced Δψm loss, whereas the broad-range inhibitor of cathepsins (Cathepsin Inhibitor I) could suppress the LAPTM5-induced apoptotic sub-G1 peak and Δψm loss, by ~22% and ~23%, respectively, suggesting that the LAPTM5-mediated Δψm loss was exerted at least in part in a cathepsin-dependent manner. Together, these results demonstrate that ectopic overexpression of LAPTM5 in HeLa cells induced apoptosis via cleavage of Mcl-1 and Bid by a LAPTM5-associated lysosomal pathway, and subsequent activation of the mitochondria-dependent caspase cascade. PMID:28464033

miR-124 radiosensitizes human esophageal cancer cell TE-1 by targeting CDK4.

PubMed

Zhang, Y H; Wang, Q Q; Li, H; Ye, T; Gao, F; Liu, Y C

2016-06-03

Radiotherapy is one of the most important treatments for esophageal cancer, but radioresistance remains a major challenge. Previous studies have shown that microRNAs (miRNAs or miRs) are involved in human cancers. miR-124 has been widely reported in various cancers and it is intimately involved in proliferation, cell cycle regulation, apoptosis, migration, and invasion of cancer cells. The aim of this study was to explore the relationship between the miR-124/cyclin-dependent kinase 4 (CDK4) axis and the radiosensitivity of esophageal cancer cells. In this study, we identified the reduced expression of miR-124 in 18 paired esophageal cancer tissues compared to their matched normal tissues. In order to investigate the physiological role of miR-124 in esophageal cancer, the cell counting kit-8 (CCK-8) assay and wound healing assay were performed, and the results suggest that miR-124 overexpression decreases tumor growth and aggression. Next, we detected the effects of ectopic miR-124 expression on the apoptosis of an esophageal cancer cell line (TE-1) following radiotherapy. Using the CCK-8 assay and Hoechst 332528 stain, we found that ectopic expression of miR-124 led to a higher percentage of apoptotic cells. Finally, we identified that CDK4 is a direct target of miR-124 in TE-1 cells using target prediction algorithms and a luciferase reporter assay. Moreover, western blot assay confirmed that CDK4 was downregulated during miR-124 transfection. Taken together, we illustrate that the miR-124/CDK4 axis plays an important role in radiation sensitivity of human esophageal cancer cells by targeting CDK4.

Nucleoprotein of influenza A virus negatively impacts antiapoptotic protein API5 to enhance E2F1-dependent apoptosis and virus replication.

PubMed

Mayank, A K; Sharma, S; Nailwal, H; Lal, S K

2015-12-17

Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.

Mitochondrial targeting of HIF-1α inhibits hypoxia-induced apoptosis independently of its transcriptional activity.

PubMed

Li, Hong-Sheng; Zhou, Yan-Ni; Li, Lu; Li, Sheng-Fu; Long, Dan; Chen, Xue-Lu; Zhang, Jia-Bi; Li, You-Ping; Feng, Li

2018-04-25

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates adaptive responses to hypoxia by nuclear translocation and regulation of gene expression. Mitochondrial changes are critical for the adaptive response to hypoxia. However, the transcriptional and non-transcriptional mechanisms by which HIF-1α regulates mitochondria under hypoxia are poorly understood. Here, we examined the subcellular localization of HIF-1α in human cells and identified a small fraction of HIF-1α that translocated to the mitochondria after exposure to hypoxia or hypoxia-mimicking pharmacological agents. To probe the function of this HIF-1α population, we ectopically expressed a mitochondrial-targeted form of HIF-1α (mito-HIF-1α). Expression of mito-HIF-1α was sufficient to attenuate apoptosis induced by exposure to hypoxia or H 2 O 2 -induced oxidative stress. Moreover, mito-HIF-1α expression reduced the production of reactive oxygen species, the collapse of mitochondrial membrane potential, and the expression of mitochondrial DNA-encoded mRNA in response to hypoxia. However, these functions of mito-HIF-1α were independent of its conventional transcriptional activity. Finally, the livers of mice with CCl 4 -induced fibrosis showed a progressive increase in HIF-1α association with the mitochondria, indicating the clinical relevance of this finding. These data suggested that mitochondrial HIF-1α protects against apoptosis independently of its well-known role as a transcription factor. Copyright © 2018. Published by Elsevier Inc.

The Aryl Hydrocarbon Receptor Binds to E2F1 and Inhibits E2F1-induced Apoptosis

PubMed Central

Marlowe, Jennifer L.; Fan, Yunxia; Chang, Xiaoqing; Peng, Li; Knudsen, Erik S.; Xia, Ying

2008-01-01

Cellular stress by DNA damage induces checkpoint kinase-2 (CHK2)-mediated phosphorylation and stabilization of the E2F1 transcription factor, leading to induction of apoptosis by activation of a subset of proapoptotic E2F1 target genes, including Apaf1 and p73. This report characterizes an interaction between the aryl hydrocarbon (Ah) receptor (AHR), a ligand-activated transcription factor, and E2F1 that results in the attenuation of E2F1-mediated apoptosis. In Ahr−/− fibroblasts stably transfected with a doxycycline-regulated AHR expression vector, inhibition of AHR expression causes a significant elevation of oxidative stress, γH2A.X histone phosphorylation, and E2F1-dependent apoptosis, which can be blocked by small interfering RNA-mediated knockdown of E2F1 expression. In contrast, ligand-dependent AHR activation protects these cells from etoposide-induced cell death. In cells expressing both proteins, AHR and E2F1 interact independently of the retinoblastoma protein (RB), because AHR and E2F1 coimmunoprecipitate from extracts of RB-negative cells. Additionally, chromatin immunoprecipitation assays indicate that AHR and E2F1 bind to the Apaf1 promoter at a region containing a consensus E2F1 binding site but no AHR binding sites. AHR activation represses Apaf1 and TAp73 mRNA induction by a constitutively active CHK2 expression vector. Furthermore, AHR overexpression blocks the transcriptional induction of Apaf1 and p73 and the accumulation of sub-G0/G1 cells resulting from ectopic overexpression of E2F1. These results point to a proproliferative, antiapoptotic function of the Ah receptor that likely plays a role in tumor progression. PMID:18524851

MALAT1-miR-124-RBG2 axis is involved in growth and invasion of HR-HPV-positive cervical cancer cells.

PubMed

Liu, Shikai; Song, Lili; Zeng, Saitian; Zhang, Liang

2016-01-01

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT 1) is a large, infrequently spliced non-coding RNA aberrantly expressed in cervical cancer. But the molecular mechanisms of its oncogenic role are still not quite clear. The present study explored whether there is a competing endogenous RNAs (ceRNAs) mechanism involved in the oncogenic effect of MALAT1. MALAT1 expression was firstly verified in high-risk human papillomavirus (HR-HPV)-positive tumor tissues and cell lines. Its regulation over miR-124 and the downstream target of miR-124 in regulation of growth, invasion, and apoptosis of the cancer cells are also studied. Findings of this study confirmed higher MALAT1 expression in HR-HPV (+) cervical cancer. Knockdown of endogenous MALAT1 significantly reduced cell growth rate and invasion and increased cell apoptosis of Hela and siHa cells. Besides, knockdown of MALAT1 increased the expression of miRNA-124, while ectopic expression of miR-124 decreased MALAT1 expression. In addition, we also verified a direct interaction between miR-124 and 3'UTR of GRB2. MALAT1 can indirectly modulate GRB2 expression via competing miR-124. Knockdown of GRB2 reduced cell invasion and increased cell apoptosis. In conclusion, MALAT1 can promote HR-HPV (+) cancer cell growth and invasion at least partially through the MALAT1-miR-124-RBG2 axis. This finding might provide some useful evidence about the lncRNA interaction regulatory network in tumorigenesis cervical cancer.

CMTM5 exhibits tumor suppressor activity through promoter methylation in oral squamous cell carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Heyu; Nan, Xu; Li, Xuefen

Highlights: • Down-regulation of CMTM5 expression in OSCC tissues was found. • The promoter methylation status of CMTM5 was measured. • CMTM5-v1 inhibited cell proliferation and migration and induced apoptosis. • CMTM5 might act as a putative tumor suppressor gene in OSCC. - Abstract: Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancies in the head and neck region. CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) has been recently implicated as a tumor suppressor gene in several cancer types. Herein, we examined the expression and function of CMTM5 in oral squamous cell carcinoma. CMTM5 wasmore » down-regulated in oral squamous cell lines and tumor samples from patients with promoter methylation. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored CMTM5 expression. In the OSCC cell lines CAL27 and GNM, the ectopic expression of CMTM5-v1 strongly inhibited cell proliferation and migration and induced apoptosis. In addition, CMTM5-v1 inhibited tumor formation in vivo. Therefore, CMTM5 might act as a putative tumor suppressor gene through promoter methylation in oral squamous cell carcinoma.« less

Expression of Death Receptor 4 Is Positively Regulated by MEK/ERK/AP-1 Signaling and Suppressed upon MEK Inhibition*

PubMed Central

Yao, Weilong; Oh, You-Take; Deng, Jiusheng; Yue, Ping; Deng, Liang; Huang, Henry; Zhou, Wei; Sun, Shi-Yong

2016-01-01

Death receptor 4 (DR4) is a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and triggers apoptosis upon ligation with TRAIL or aggregation. MEK/ERK signaling is a well known and the best-studied effector pathway downstream of Ras and Raf. This study focuses on determining the impact of pharmacological MEK inhibition on DR4 expression and elucidating the underlying mechanism. We found that several MEK inhibitors including MEK162, AZD6244, and PD0325901 effectively decreased DR4 protein levels including cell surface DR4 in different cancer cell lines. Accordingly, pre-treatment of TRAIL-sensitive cancer cell lines with a MEK inhibitor desensitized them to TRAIL-induced apoptosis. These results indicate that MEK inhibition negatively regulates DR4 expression and cell response to TRAIL-induced apoptosis. MEK inhibitors did not alter DR4 protein stability, rather decreased its mRNA levels, suggesting a transcriptional regulation. In contrast, enforced activation of MEK/ERK signaling by expressing ectopic B-Raf (V600E) or constitutively activated MEK1 (MEK1-CA) or MEK2 (MEK2-CA) activated ERK and increased DR4 expression; these effects were inhibited when a MEK inhibitor was present. Promoter analysis through deletion and mutation identified the AP-1 binding site as an essential response element for enhancing DR4 transactivation by MEK1-CA. Furthermore, inhibition of AP-1 by c-Jun knockdown abrogated the ability of MEK1-CA to increase DR4 promoter activity and DR4 expression. These results suggest an essential role of AP-1 in mediating MEK/ERK activation-induced DR4 expression. Our findings together highlight a previously undiscovered mechanism that positively regulates DR4 expression through activation of the MEK/ERK/AP-1 signaling pathway. PMID:27576686

Upregulation of the long non-coding RNA SNHG1 predicts poor prognosis, promotes cell proliferation and invasion, and reduces apoptosis in glioma.

PubMed

Wang, Qiang; Li, Qing; Zhou, Peng; Deng, Danni; Xue, Lian; Shao, Naiyuan; Peng, Ya; Zhi, Feng

2017-07-01

Long non-coding RNAs (lncRNAs), which are non-coding RNAs with a length above 200 nucleotides, have emerged as novel and important gene expression modulators in carcinogenesis. Recent evidence indicates that the lncRNA small nucleolar RNA host gene 1 (SNHG1) functions as an oncogene in several types of human cancers. However, its function in the development of glioma remains unknown. The aim of this research was to investigate the clinical aspects and biological mechanisms of SNHG1 in glioma. SNHG1 expression was measured in glioma tissues and cell lines by quantitative real-time PCR (qRT-PCR). The association between SNHG1 expression in tissues and clinicopathological characteristics and prognosis in glioma patients was also explored. Gain-of-function and loss-of-function studies using SNHG1 cDNA and siRNA, respectively, were used to investigate the role of SNHG1 in cell proliferation, invasion and apoptosis in glioma. SNHG1 was highly expressed in glioma tissues, and its upregulation was closely related to old age. Kaplan-Meier analysis showed that high expression of SNHG1 was significantly associated with poor overall survival (OS). Functionally, ectopic expression of SNHG1 enhanced cell proliferation and cell invasion and reduced cell apoptosis in vitro, while SNHG1 knockdown reversed these effects. Taken together, our findings indicate that SNHG1 functions as an oncogene in glioma and may serve as a novel therapeutic target in future treatments. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Distinct p300-Responsive Mechanisms Promote Caspase-Dependent Apoptosis by Human T-Cell Lymphotropic Virus Type 1 Tax Protein

PubMed Central

Nicot, Christophe; Harrod, Robert

2000-01-01

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-dl-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-κB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia. PMID:11046153

Distinct p300-responsive mechanisms promote caspase-dependent apoptosis by human T-cell lymphotropic virus type 1 Tax protein.

PubMed

Nicot, C; Harrod, R

2000-11-01

The dysregulation of cellular apoptosis pathways has emerged as a critical early event associated with the development of many types of human cancers. Numerous viral and cellular oncogenes, aside from their inherent transforming properties, are known to induce programmed cell death, consistent with the hypothesis that genetic defects are required to support tumor survival. Here, we report that nuclear expression of the CREB-binding protein (CBP)/p300-binding domain of the human T-cell lymphotropic virus type 1 (HTLV-1) transactivator, Tax, triggers an apoptotic death-inducing signal during short-term clonal analyses, as well as in transient cell death assays. Coexpression of the antiapoptotic factor Bcl-2 increased serum stimulation; incubation with the chemical caspase inhibitor z-Val-Ala-DL-Asp fluoromethylketone antagonized Tax-induced cell death. The CBP/p300-binding defective Tax mutants K88A and V89A exhibited markedly reduced cytotoxic effects compared to the wild-type Tax protein. Importantly, nuclear expression of the minimal CBP/p300-binding peptide of Tax induced apoptosis in the absence of Tax-dependent transcriptional activities, while its K88A counterpart did not cause cell death. Further, Tax-mediated apoptosis was effectively prevented by ectopic expression of the p300 coactivator. We also report that activation of the NF-kappaB transcription pathway by Tax, under growth arrest conditions, results in apoptosis that occurs independent of direct Tax coactivator effects. Our results allude to a novel pivotal role for the transcriptional coactivator p300 in determining cell fate and raise the possibility that dysregulated coactivator usage may pose an early barrier to transformation that must be selectively overcome as a prerequisite for the initiation of neoplasia.

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

PubMed

Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2004-05-01

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.

MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

PubMed Central

2010-01-01

Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K) pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer. PMID:20409325

Id-1 promotes osteosarcoma cell growth and inhibits cell apoptosis via PI3K/AKT signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Liang; Liao, Qi; Tang, Qiang

2016-02-12

Accumulating evidence reveals that Id-1 is upregulated and functions as a potential tumor promoter in several human cancer types. However, the role of Id-1 in osteosarcoma (OS) is unknown. In present study, we found that Id-1 expression was elevated in OS tissues than adjacent normal bone tissues. More importantly, we demonstrated that overexpression of Id-1 is significantly correlated with tumor progression and poor survival in OS patients. Furthermore, increased expression of Id-1 was observed in OS cell lines and ectopic expression of Id-1 significantly enhanced in vitro cell proliferation and promoted in vivo tumor growth, whereas knockdown of Id-1 suppressed OS cellsmore » growth. Moreover, our experimental data revealed that Id-1 promotes cell proliferation by facilitating cell cycle progression and inhibits cell apoptosis. Mechanistically, the effects of Id-1 in OS cells is at least partly through activation of PI3K/Akt signaling pathway. Therefore, we identified a tumorigenic role of Id-1 in OS and suggested a potential therapeutic target for OS patients. - Highlights: • Id-1 expression is positively correlated in OS patients with poor prognosis. • Overexpression of Id-1 promotes OS cell growth in vitro and in vivo. • Id-1induces cell cycle progression and inhibits cell apoptosis. • PI3K/Akt signaling pathway contributed to the oncogenic effects of Id-1 in OS cells.« less

Systems-level effects of ectopic galectin-7 reconstitution in cervical cancer and its microenvironment.

PubMed

Higareda-Almaraz, Juan Carlos; Ruiz-Moreno, Juan S; Klimentova, Jana; Barbieri, Daniela; Salvador-Gallego, Raquel; Ly, Regina; Valtierra-Gutierrez, Ilse A; Dinsart, Christiane; Rabinovich, Gabriel A; Stulik, Jiri; Rösl, Frank; Rincon-Orozco, Bladimiro

2016-08-24

Galectin-7 (Gal-7) is negatively regulated in cervical cancer, and appears to be a link between the apoptotic response triggered by cancer and the anti-tumoral activity of the immune system. Our understanding of how cervical cancer cells and their molecular networks adapt in response to the expression of Gal-7 remains limited. Meta-analysis of Gal-7 expression was conducted in three cervical cancer cohort studies and TCGA. In silico prediction and bisulfite sequencing were performed to inquire epigenetic alterations. To study the effect of Gal-7 on cervical cancer, we ectopically re-expressed it in the HeLa and SiHa cervical cancer cell lines, and analyzed their transcriptome and SILAC-based proteome. We also examined the tumor and microenvironment host cell transcriptomes after xenotransplantation into immunocompromised mice. Differences between samples were assessed with the Kruskall-Wallis, Dunn's Multiple Comparison and T tests. Kaplan-Meier and log-rank tests were used to determine overall survival. Gal-7 was constantly downregulated in our meta-analysis (p < 0.0001). Tumors with combined high Gal-7 and low galectin-1 expression (p = 0.0001) presented significantly better prognoses (p = 0.005). In silico and bisulfite sequencing assays showed de novo methylation in the Gal-7 promoter and first intron. Cells re-expressing Gal-7 showed a high apoptosis ratio (p < 0.05) and their xenografts displayed strong growth retardation (p < 0.001). Multiple gene modules and transcriptional regulators were modulated in response to Gal-7 reconstitution, both in cervical cancer cells and their microenvironments (FDR < 0.05 %). Most of these genes and modules were associated with tissue morphogenesis, metabolism, transport, chemokine activity, and immune response. These functional modules could exert the same effects in vitro and in vivo, even despite different compositions between HeLa and SiHa samples. Gal-7 re-expression affects the regulation of molecular networks in cervical cancer that are involved in diverse cancer hallmarks, such as metabolism, growth control, invasion and evasion of apoptosis. The effect of Gal-7 extends to the microenvironment, where networks involved in its configuration and in immune surveillance are particularly affected.

HOXB1 Is a Tumor Suppressor Gene Regulated by miR-3175 in Glioma

PubMed Central

Han, Liang; Liu, Dehua; Li, Zhaohui; Tian, Nan; Han, Ziwu; Wang, Guang; Fu, Yao; Guo, Zhigang; Zhu, Zifeng

2015-01-01

The HOXB1 gene plays a critical role as an oncogene in diverse tumors. However, the functional role of HOXB1 and the mechanism regulating HOXB1 expression in glioma are not fully understood. A preliminary bioinformatics analysis showed that HOXB1 is ectopically expressed in glioma, and that HOXB1 is a possible target of miR-3175. In this study, we investigated the function of HOXB1 and the relationship between HOXB1 and miR-3175 in glioma. We show that HOXB1 expression is significantly downregulated in glioma tissues and cell lines, and that its expression may be closely associated with the degree of malignancy. Reduced HOXB1 expression promoted the proliferation and invasion of glioma cells, and inhibited their apoptosis in vitro, and the downregulation of HOXB1 was also associated with worse survival in glioma patients. More importantly, HOXB1 was shown experimentally to be a direct target of miR-3175 in this study. The downregulated expression of miR-3175 inhibited cell proliferation and invasion, and promoted apoptosis in glioma. The oncogenicity induced by low HOXB1 expression was prevented by an miR-3175 inhibitor in glioma cells. Our results suggest that HOXB1 functions as a tumor suppressor, regulated by miR-3175 in glioma. These results clarify the pathogenesis of glioma and offer a potential target for its treatment. PMID:26565624

Inhibition of Ced-3/ICE-related Proteases Does Not Prevent Cell Death Induced by Oncogenes, DNA Damage, or the Bcl-2 Homologue Bak

PubMed Central

McCarthy, Nicola J.; Whyte, Moira K.B.; Gilbert, Christopher S.; Evan, Gerard I.

1997-01-01

There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1β– converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode “death” gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage–induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing. PMID:9008715

Targeting Phosphatidylinositide3-Kinase/Akt pathway by BKM120 for radiosensitization in hepatocellular carcinoma

PubMed Central

Liu, Wei-Lin; Gao, Ming; Tzen, Kai-Yuan; Tsai, Chiao-Ling; Hsu, Feng-Ming; Cheng, Ann-Lii; Cheng, Jason Chia-Hsien

2014-01-01

Tumor control of hepatocellular carcinoma by radiotherapy remains unsatisfactory. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in inhibiting cancer cell death. Elevated PI3K/Akt activity is associated with increased cellular resistance to irradiation. Our aim was to determine whether the inhibition of PI3K/Akt activity by a PI3K inhibitor, BKM120, contributes to the increased sensitivity of liver cancer cells to irradiation. The hepatocellular carcinoma cell lines (Huh7 and BNL) were used to evaluate the in vitro synergism between BKM120 and irradiation. Balb/c mice bearing ectopic BNL xenografts were treated with BKM120 and/or radiotherapy to assess the in vivo response. BKM120 increased cell killing by radiation, increased the expression of apoptotic markers, and suppressed the repair of radiation-induced DNA double-strand breaks. BKM120 pretreatment inhibited radiation-induced Akt phosphorylation and enhanced the tumor-suppressive effect and radiation-induced tumor cell apoptosis in ectopic xenografts. Inhibition of mTOR phosphorylation by rapamycin enhanced the radiosensitivity of BKM120-treated hepatocellular carcinoma cells. The synergism between BKM120 and irradiation likely inhibits the activation of Akt by radiation, leading to increased cell apoptosis and suppression of DNA-double-strand breaks repair in hepatocellular carcinoma cells. These data suggest that the BKM120/radiation combination may be a strategy worthy of clinical trials. PMID:25004403

Circular RNA HIPK3 promotes gallbladder cancer cell growth by sponging microRNA-124.

PubMed

Kai, Ding; Yannian, Liao; Yitian, Chen; Dinghao, Gong; Xin, Zhao; Wu, Ji

2018-06-18

Recent studies have implied that circHIPK3, an abundant circular RNA (circRNA), participates in tumorigenesis and cancer progression. Its expression and potential functions in human gallbladder cancer were examined in this study. We show that circHIPK3 is upregulated in human gallbladder cancer cells. But its level is low in gallbladder epithelial cells. circHIPK3 silencing by targeted siRNA potently inhibited survival and proliferation of established and primary human gallbladder cancer cells, while inducing cell apoptosis. Conversely, ectopic over-expression of circHIPK3 can further promote cancer cell proliferation. In gallbladder cancer cells, circHIPK3 sponged the tumor-suppressive microRNA-124 (miR-124) to sequester and inhibit its activity, thereby leading to increased expression of miR-124 targets, including ROCK1 (rho-associated protein kinase 1) and CDK6 (rho-associated protein kinase). Ectopic over-expression of miR-124 b y a lentiviral vector mimicked and abolished actions by circHIPK3 siRNA in gallbladder cancer cells. At last, we show that circHIPK3 is upregulated in human gallbladder cancer tissues, which is correlated with miR-124 downregulation and ROCK1-CDK6 upregulation. Together, we conclude that circHIPK3 promotes gallbladder cancer cell growth possibly by sponging miR-124. The over-expressed circHIPK3 could be a novel therapeutic target and diagnosis marker of human gallbladder cancer. Copyright © 2018. Published by Elsevier Inc.

Autophagy-mediated degradation of IAPs and c-FLIP L potentiates apoptosis induced by combination of TRAIL and Chal-24

DOE PAGES

Xu, Jennings; Xu, Xiuling; Shi, Shaoqing; ...

2015-11-02

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. In this paper, we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIPL andmore » c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by Chal-24 and TRAIL combination, which was associated with attenuation of c-FLIPL and c-IAPs degradation. In conclusion, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIPL and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.« less

Cancerous inhibitor of protein phosphatase 2A determines bortezomib-induced apoptosis in leukemia cells

PubMed Central

Liu, Chun-Yu; Shiau, Chung-Wai; Kuo, Hsin-Yu; Huang, Hsiang-Po; Chen, Ming-Huang; Tzeng, Cheng-Hwai; Chen, Kuen-Feng

2013-01-01

The multiple cellular targets affected by proteasome inhibition implicate a potential role for bortezomib, a first-in-class proteasome inhibitor, in enhancing antitumor activities in hematologic malignancies. Here, we examined the antitumor activity and drug targets of bortezomib in leukemia cells. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis assays and associated molecular events assessed by Western Blot. Gene silencing was performed by small interference RNA. Drug was tested in vivo in xenograft models of human leukemia cell lines and in primary leukemia cells. Clinical samples were assessed by immunohistochemical staining. Bortezomib differentially induced apoptosis in leukemia cells that was independent of its proteasome inhibition. Cancerous inhibitor of protein phosphatase 2A, a cellular inhibitor of protein phosphatase 2A, mediated the apoptotic effect of bortezomib. Bortezomib increased protein phosphatase 2A activity in sensitive leukemia cells (HL-60 and KG-1), but not in resistant cells (MOLT-3 and K562). Bortezomib’s downregulation of cancerous inhibitor of protein phosphatase 2A and phospho-Akt correlated with its drug sensitivity. Furthermore, cancerous inhibitor of protein phosphatase 2A negatively regulated protein phosphatase 2A activity. Ectopic expression of CIP2A up-regulated phospho-Akt and protected HL-60 cells from bortezomib-induced apoptosis, whereas silencing CIP2A overcame the resistance to bortezomib-induced apoptosis in MOLT3 and K562 cells. Importantly, bortezomib exerted in vivo antitumor activity in HL-60 xenografted tumors and induced cell death in some primary leukemic cells. Cancerous inhibitor of protein phosphatase 2A was expressed in leukemic blasts from bone marrow samples. Cancerous inhibitor of protein phosphatase 2A plays a major role in mediating bortezomib-induced apoptosis in leukemia cells. PMID:22983581

Misexpression of cyclin B3 leads to aberrant spermatogenesis.

PubMed

Refik-Rogers, Jale; Manova, Katia; Koff, Andrew

2006-09-01

Mus musculus cyclin B3 is an early meiotic cyclin that is expressed in leptotene and zygotene phases during gametogenesis. In order to determine whether downregulation of cyclin B3 at zygotene-pachytene transition was important for normal spermatogenesis, we investigated the consequences of expressing H. sapiens cyclin B3 after zygotene in mouse testes. Prolonging expression of cyclin B3 until the end of meiosis led to a reduction in sperm counts and disruption of spermatogenesis in four independent lines of transgenic mice. There were three distinct morphological defects associated with the ectopic expression of cyclin B3. Seminiferous tubules were either depleted of germ cells, had an abnormal cell mass in the lumen, or were characterized by the presence of abnormal round spermatids. These defects were associated with increased apoptosis in the testes. These results suggest that downregulation of cyclin B3 at the zygotene-pachytene transition is required to ensure normal spermatogenesis.

The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

PubMed

Ren, Hui; Koo, Junghui; Guan, Baoxiang; Yue, Ping; Deng, Xingming; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2013-11-22

The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis

PubMed Central

2013-01-01

Background The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. Results API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. Conclusions API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis. PMID:24261825

Differential participation of angiotensin II type 1 and 2 receptors in the regulation of cardiac cell death triggered by angiotensin II.

PubMed

Aránguiz-Urroz, Pablo; Soto, Dagoberto; Contreras, Ariel; Troncoso, Rodrigo; Chiong, Mario; Montenegro, José; Venegas, Daniel; Smolic, Christian; Ayala, Pedro; Thomas, Walter G; Lavandero, Sergio; Díaz-Araya, Guillermo

2009-05-01

The Angiotensin II (Ang II) type 1 (AT(1)R) and type 2 (AT(2)R) receptors are increased in the heart following myocardial infarction and dilated cardiomyopathy, yet their contribution at a cellular level to compensation and/or failure remains controversial. We ectopically expressed AT(1)R and AT(2)R in cultured adult rat cardiomyocytes and cardiac fibroblasts to investigate Ang II-mediated cardiomyocyte hypertrophy and cardiac cell viability. In adult rat cardiomyocytes, Ang II did not induce hypertrophy via the AT(1)R, and no effect of Ang II on cell viability was observed following AT(1)R or AT(2)R expression. In adult rat cardiac fibroblasts, Ang II stimulated cell death by apoptosis via the AT(1)R (but not the AT(2)R), which required the presence of extracellular calcium, and induced a rapid dissipation of mitochondrial membrane potential, which was significant from 8 h. We conclude that Ang II/AT(1)R triggers apoptosis in adult rat cardiac fibroblasts, which is dependent on Ca2+ influx.

Hsa-miR-134 suppresses non-small cell lung cancer (NSCLC) development through down-regulation of CCND1

PubMed Central

Sun, Cheng-Cao; Li, Shu-Jun; Li, De-Jia

2016-01-01

Hsa-miRNA-134 (miR-134) has recently been discovered to have anticancer efficacy in different organs. However, the role of miR-134 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-134 on the development of NSCLC. The results indicated that miR-134 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-134 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-134 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-134 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-134, which was inversely correlated with miR-134 expression in NSCLC. Taken together, our results demonstrated that miR-134 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1. PMID:27166267

Hsa-miR-326 targets CCND1 and inhibits non-small cell lung cancer development

PubMed Central

Li, Shujun; Yang, Cuili; Xi, Yongyong; Wang, Liang; Zhang, Feng; Fu, Yunfeng; Li, Dejia

2016-01-01

Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1. PMID:26840018

Carfilzomib induces leukaemia cell apoptosis via inhibiting ELK1/KIAA1524 (Elk-1/CIP2A) and activating PP2A not related to proteasome inhibition.

PubMed

Liu, Chun-Yu; Hsieh, Feng-Shu; Chu, Pei-Yi; Tsai, Wen-Chun; Huang, Chun-Teng; Yu, Yuan-Bin; Huang, Tzu-Ting; Ko, Po-Shen; Hung, Man-Hsin; Wang, Wan-Lun; Shiau, Chung-Wai; Chen, Kuen-Feng

2017-06-01

Enhancing the tumour suppressive activity of protein phosphatase 2A (PP2A) has been suggested to be an anti-leukaemic strategy. KIAA1524 (also termed CIP2A), an oncoprotein inhibiting PP2A, is associated with disease progression in chronic myeloid leukaemia and may be prognostic in cytogenetically normal acute myeloid leukaemia. Here we demonstrated that the selective proteasome inhibitor, carfilzomib, induced apoptosis in sensitive primary leukaemia cells and in sensitive leukaemia cell lines, associated with KIAA1524 protein downregulation, increased PP2A activity and decreased p-Akt, but not with the proteasome inhibition effect of carfilzomib. Ectopic expression of KIAA1524, or pretreatment with the PP2A inhibitor, okadaic acid, suppressed carfilzomib-induced apoptosis and KIAA1524 downregulation in sensitive cells, whereas co-treatment with the PP2A agonist, forskolin, enhanced carfilzomib-induced apoptosis in resistant cells. Mechanistically, carfilzomib affected KIAA1524 transcription through disturbing ELK1 (Elk-1) binding to the KIAA1524 promoter. Moreover, the drug sensitivity and mechanism of carfilzomib in xenograft mouse models correlated well with the effects of carfilzomib on KIAA1524 and p-Akt expression, as well as PP2A activity. Our data disclosed a novel drug mechanism of carfilzomib in leukaemia cells and suggests the potential therapeutic implication of KIAA1524 in leukaemia treatment. © 2017 John Wiley & Sons Ltd.

Cellular glucose-6-phosphate dehydrogenase (G6PD) status modulates the effects of nitric oxide (NO) on human foreskin fibroblasts.

PubMed

Cheng, M L; Ho, H Y; Liang, C M; Chou, Y H; Stern, A; Lu, F J; Chiu, D T

2000-06-23

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in cellular redox homeostasis, which is crucial for cell survival. In the present study, we found that G6PD status determines the response of cells exposed to nitric oxide (NO) donor. Treatment with NO donor, sodium nitroprusside (SNP), caused apoptosis in G6PD-deficient human foreskin fibroblasts (HFF1), whereas it was growth stimulatory in the normal counterpart (HFF3). Such effects were abolished by NO scavengers like hemoglobin. Ectopic expression of G6PD in HFF1 cells switched the cellular response to NO from apoptosis to growth stimulation. Experiments with 1H-¿1,2,4ŏxadiazolo¿4, 3-aquinoxalin-1-one and 8-bromo-cGMP showed that the effects of NO on HFF1 and HFF3 cells were independent of cGMP signalling pathway. Intriguingly, trolox prevented the SNP-induced apoptosis in HFF1 cells. These data demonstrate that G6PD plays a critical role in regulation of cell growth and survival.

Id-1 activation of PI3K/Akt/NFkappaB signaling pathway and its significance in promoting survival of esophageal cancer cells.

PubMed

Li, Bin; Cheung, Pak Yan; Wang, Xianghong; Tsao, Sai Wah; Ling, Ming Tat; Wong, Yong Chuan; Cheung, Annie L M

2007-11-01

Inhibitor of differentiation or DNA binding (Id-1) is a helix-loop-helix protein that is over-expressed in many types of cancer including esophageal cancer. This study aims to investigate its effects on the phosphatidylinositol-3-kinase (PI3K)/Akt/ nuclear factor kappa B (NFkappaB) signaling pathway and the significance in protecting esophageal cancer cells against apoptosis. We found elevated expression of phosphorylated forms of Akt, glycogen synthase kinase 3beta and inhibitor of kappa B, as well as increased nuclear translocation of NFkappaB subunit p65 and NFkappaB DNA-binding activity, in esophageal cancer cells with stable ectopic Id-1 expression. Transient transfection of Id-1 into HEK293 cells confirmed activation of PI3K/Akt/NFkappaB signaling and the effects were counteracted by the PI3K inhibitor LY294002. Treatment with tumor necrosis factor-alpha (TNF-alpha) elicited a significantly weaker apoptotic response, following a marked and sustained activation of Akt and NFkappaB in the Id-1-over-expressing cells, compared with the vector control. The effects of Id-1 on the PI3K/Akt/NFkappaB signaling pathway and apoptosis were reversed in esophageal cancer cells transfected with siRNA against Id-1. In addition, inhibition of PI3K or NFkappaB signaling using the PI3K inhibitor LY294002 or the NFkappaB inhibitor Bay11-7082 increased the sensitivity of Id-1-over-expressing esophageal cancer cells to TNF-alpha-induced apoptosis. Our results provide the first evidence that Id-1 induces the activation of PI3K/Akt/NFkappaB signaling pathway, and protects esophageal cancer cells from TNF-alpha-induced apoptosis in vitro. Inactivation of Id-1 may provide us with a novel strategy to improve the treatment and survival of patients with esophageal cancer.

Ataxin-1 Poly(Q)-induced Proteotoxic Stress and Apoptosis Are Attenuated in Neural Cells by Docosahexaenoic Acid-derived Neuroprotectin D1*

PubMed Central

Calandria, Jorgelina M.; Mukherjee, Pranab K.; de Rivero Vaccari, Juan Carlos; Zhu, Min; Petasis, Nicos A.; Bazan, Nicolas G.

2012-01-01

Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways. PMID:22511762

FAS-antisense 1 lncRNA and production of soluble versus membrane Fas in B-cell lymphoma

PubMed Central

Sehgal, Lalit; Mathur, Rohit; Braun, Frank K.; Wise, Jillian F.; Berkova, Zuzana; Neelapu, Sattva; Kwak, Larry W.; Samaniego, Felipe

2018-01-01

Impaired Fas-mediated apoptosis is associated with poor clinical outcomes and cancer chemoresistance. Soluble Fas receptor (sFas), produced by skipping of exon 6, inhibits apoptosis by sequestering Fas ligand. Serum sFas is associated with poor prognosis of non-Hodgkin's lymphomas. We found that the alternative splicing of Fas in lymphomas is tightly regulated by a lncRNA corresponding to an antisense transcript of Fas (FAS-AS1). Levels of FAS-AS1 correlate inversely with production of sFas and FAS-AS1 binding to the RBM5 inhibits RBM5-mediated exon 6 skipping. EZH2, often mutated or overexpressed in lymphomas, hyper-methylates the FAS-AS1 promoter and represses the FAS-AS1 expression. EZH2-mediated repression of FAS-AS1 promoter can be released by DZNeP or overcome by ectopic expression of FAS-AS1, both of which increase levels of FAS-AS1 and correspondingly decrease expression of sFas. Treatment with Bruton’s tyrosine kinase (BTK) inhibitor or EZH2 knockdown decreases the levels of EZH2, RBM5 and sFas thereby enhances Fas-mediated apoptosis. This is the first report showing functional regulation of Fas repression by its antisense RNA. Our results reveal new therapeutic targets in lymphomas and provide a rationale for the use of EZH2 inhibitors or ibrutinib in combination with chemotherapeutic agents that recruit Fas for effective cell killing. PMID:24811343

THAP5 is a human cardiac-specific inhibitor of cell cycle that is cleaved by the proapoptotic Omi/HtrA2 protease during cell death.

PubMed

Balakrishnan, Meenakshi P; Cilenti, Lucia; Mashak, Zineb; Popat, Paiyal; Alnemri, Emad S; Zervos, Antonis S

2009-08-01

Omi/HtrA2 is a mitochondrial serine protease that has a dual function: while confined in the mitochondria, it promotes cell survival, but when released into the cytoplasm, it participates in caspase-dependent as well as caspase-independent cell death. To investigate the mechanism of Omi/HtrA2's function, we set out to isolate and characterize novel substrates for this protease. We have identified Thanatos-associated protein 5 (THAP5) as a specific interactor and substrate of Omi/HtrA2 in cells undergoing apoptosis. This protein is an uncharacterized member of the THAP family of proteins. THAP5 has a unique pattern of expression and is found predominantly in the human heart, although a very low expression is also seen in the human brain and muscle. THAP5 protein is localized in the nucleus and, when ectopically expressed, induces cell cycle arrest. During apoptosis, THAP5 protein is degraded, and this process can be blocked using a specific Omi/HtrA2 inhibitor, leading to reduced cell death. In patients with coronary artery disease, THAP5 protein levels substantially decrease in the myocardial infarction area, suggesting a potential role of this protein in human heart disease. This work identifies human THAP5 as a cardiac-specific nuclear protein that controls cell cycle progression. Furthermore, during apoptosis, THAP5 is cleaved and removed by the proapoptotic Omi/HtrA2 protease. Taken together, we provide evidence to support that THAP5 and its regulation by Omi/HtrA2 provide a new link between cell cycle control and apoptosis in cardiomyocytes.

Epigenetic identification of ZNF545 as a functional tumor suppressor in multiple myeloma via activation of p53 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Yu; Zhan, Qian; Xu, Hongying

The KRAB–zinc-finger protein ZNF545 was recently identified as a potential suppressor gene in several tumors. However, the regulatory mechanisms of ZNF545 in tumorigenesis remain unclear. In this study, we investigated the expression and roles of ZNF545 in multiple myeloma (MM). ZNF545 was frequently downregulated in MM tissues compared with non-tumor bone marrow tissues. ZNF545 expression was silenced by promoter methylation in MM cell lines, and could be restored by demethylation treatment. ZNF545 methylation was detected in 28.3% of MM tissues, compared with 4.3% of normal bone marrow tissues. ZNF545 transcriptionally activated the p53 signaling pathway but had no effect onmore » Akt in MM, whereas ectopic expression of ZNF545 in silenced cells suppressed their proliferation and induced apoptosis. We therefore identified ZNF545 as a novel tumor suppressor inhibiting tumor growth through activation of the p53 pathway in MM. Moreover, tumor-specific methylation of ZNF545 may represent an epigenetic biomarker for MM diagnosis, and a potential target for specific therapy. -- Highlights: •Downregulated ZNF545 in MM tissues and cell lines and ectopic expression of ZNF545 suppresses tumor growth. •Tumor-specific methylation of ZNF545 represents an epigenetic biomarker for MM diagnosis, and a potential target for specific therapy. •ZNF545 exerts its tumor suppressive effects via transcriptional activating p53 pathway.« less

YThe BigH3 Tumor Suppressor Gene in Radiation-Induced Malignant Transformation of Human Bronchial Epithelial Cells

NASA Astrophysics Data System (ADS)

Zhao, Y.; Shao, G.; Piao, C.; Hei, T.

Carcinogenesis is a multi-stage process with sequences of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer Previous studies from this laboratory have identified a 7 fold down- regulation of the novel tumor suppressor Big-h3 among radiation induced tumorigenic BEP2D cells Furthermore ectopic re-expression of this gene suppresses tumorigenic phenotype and promotes the sensitivity of these tumor cells to etoposide-induced apoptosis To extend these studies using a genomically more stable bronchial cell line we ectopically expresses the catalytic subunit of telomerase hTERT in primary human small airway epithelial SAE cells and generated several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice These cells show no alteration in the p53 gene but a decrease in p16 expression Exponentially growing SAEh cells were exposed to graded doses of 1 GeV nucleon of 56 Fe ions accelerated at the Brookhaven National Laboratory Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium These findings indicate

Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome

PubMed Central

Ryerson, Melissa R.; Richards, Monique M.; Hawkins, Christine J.

2017-01-01

ABSTRACT Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1–procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions. IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules. PMID:28904196

PRL-3 Is Involved in Estrogen- and IL-6-Induced Migration of Endometrial Stromal Cells From Ectopic Endometrium.

PubMed

Ren, Shifan; Zhou, Yefang; Fang, Xiaoling; She, Xiaoling; Wu, Yilin; Wu, Xianqing

2016-05-24

To investigate the role of phosphatase of regenerating liver-3 (PRL-3) in the 17β-estradiol (E2)- and interleukin 6 (IL-6)-induced migration of endometrial stromal cells (ESCs) from ectopic endometrium. Ectopic endometrial tissues were collected from patients with endometriosis, and PRL-3 expression in ectopic and eutopic endometrium was examined by immunohistochemistry. Endometrial stromal cells isolated from ectopic endometrium were treated with E2, progesterone (P), IL-6, or sodium orthovanadate (Sov) to inhibit PRL-3. Total RNA and protein were extracted from ESCs after treatment for quantitative real-time polymerase chain reaction and Western blot analyses. Cell migration was assessed using a scratch wound assay. Phosphatase of regenerating liver 3 protein was highly expressed in the endometrial glandular cells (EGCs) and ESCs in ectopic endometrium, whereas its weak expression was observed only in EGCs in eutopic endometrium. Both E2 and IL-6 treatment significantly increased PRL-3 messenger RNA and protein expression, and P treatment significantly inhibited PRL-3 expression. However, E2-induced PRL-3 expression in ESCs from ectopic endometrium was significantly blocked by IL-6 antibody. Moreover, E2- and IL-6-enhanced cell migration was completely abrogated by Sov treatment. Furthermore, Sov treatment could significantly promote PTEN expression but inhibit E2- and IL-6-induced p-AKT activation. Phosphatase of regenerating liver 3 plays a key role in the E2- and IL-6-induced migration of ESCs from ectopic endometrium, a process that is involved in the PTEN-AKT signaling pathway. © The Author(s) 2016.

Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis.

PubMed

Ostrowski, Stephen M; Wright, Margaret C; Bolock, Alexa M; Geng, Xuehui; Maricich, Stephen M

2015-07-15

Merkel cells are mechanosensitive skin cells whose production requires the basic helix-loop-helix transcription factor Atoh1. We induced ectopic Atoh1 expression in the skin of transgenic mice to determine whether Atoh1 was sufficient to create additional Merkel cells. In embryos, ectopic Atoh1 expression drove ectopic expression of the Merkel cell marker keratin 8 (K8) throughout the epidermis. Epidermal Atoh1 induction in adolescent mice similarly drove widespread K8 expression in glabrous skin of the paws, but in the whisker pads and body skin ectopic K8+ cells were confined to hair follicles and absent from interfollicular regions. Ectopic K8+ cells acquired several characteristics of mature Merkel cells in a time frame similar to that seen during postnatal development of normal Merkel cells. Although ectopic K8+ cell numbers decreased over time, small numbers of these cells remained in deep regions of body skin hair follicles at 3 months post-induction. In adult mice, greater numbers of ectopic K8+ cells were created by Atoh1 induction during anagen versus telogen and following disruption of Notch signaling by conditional deletion of Rbpj in the epidermis. Our data demonstrate that Atoh1 expression is sufficient to produce new Merkel cells in the epidermis, that epidermal cell competency to respond to Atoh1 varies by skin location, developmental age and hair cycle stage, and that the Notch pathway plays a key role in limiting epidermal cell competency to respond to Atoh1 expression. © 2015. Published by The Company of Biologists Ltd.

Drosophila nemo is an essential gene involved in the regulation of programmed cell death.

PubMed

Mirkovic, Ivana; Charish, Kristi; Gorski, Sharon M; McKnight, Kristen; Verheyen, Esther M

2002-11-01

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development. Copyright 2002 Elsevier Science Ireland Ltd.

Withaferin A Suppresses Estrogen Receptor-α Expression in Human Breast Cancer Cells

PubMed Central

Hahm, Eun-Ryeong; Lee, Joomin; Huang, Yi; Singh, Shivendra V.

2011-01-01

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of MCF-7 and MDA-MB-231 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo by causing apoptosis. However, the mechanism of WA-induced apoptosis is not fully understood. The present study was designed to systematically determine the role of tumor suppressor p53 and estrogen receptor-α (ER-α) in proapoptotic response to WA using MCF-7, T47D, and ER-α overexpressing MDA-MB-231 cells as a model. WA treatment resulted in induction as well as increased Ser15 phosphorylation of p53 in MCF-7 cells, but RNA interference of this tumor suppressor gene conferred modest protection at best against WA-induced apoptosis. WA-mediated growth inhibition and apoptosis induction in MCF-7 cells were significantly attenuated in the presence of 17β-estradiol (E2). Exposure of MCF-7 cells to WA resulted in a marked decrease in protein levels of ER-α (but not ER-β) and ER-α regulated gene product pS2, and this effect was markedly attenuated in the presence of E2. WA-mediated down-regulation of ER-α protein expression correlated with a decrease in its nuclear level, suppression of its mRNA level, and inhibition of E2-dependent activation of ERE2e1b-luciferase reporter gene. Ectopic expression of ER-α in the MDA-MB-231 cell line conferred partial but statistically significant protection against WA-mediated apoptosis, but not G2/M phase cell cycle arrest. Collectively, these results indicate that WA functions as an anti-estrogen, and the proapoptotic effect of this promising natural product is partially attenuated by p53 knockdown and E2-ER-α. PMID:21432907

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment.

PubMed

Ma, Zhan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Wang, Yingzhi; Yang, Cuixia; Wang, Wenjuan; Du, Yan; Zhou, Muqing; Gao, Feng

2010-08-01

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

mda-7/IL-24 induces cell death in neuroblastoma through a novel mechanism involving AIF and ATM

PubMed Central

Bhoopathi, Praveen; Lee, Nathaniel; Pradhan, Anjan K.; Shen, Xue-Ning; Das, Swadesh K.; Sarkar, Devanand; Emdad, Luni; Fisher, Paul B.

2016-01-01

Advanced stages of neuroblastoma, the most common extracranial malignant solid tumor of the central nervous system in infants and children, are refractive to therapy. Ectopic expression of melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) promotes broad-spectrum antitumor activity in vitro, in vivo in pre-clinical animal models and in a Phase I clinical trial in patients with advanced cancers, without harming normal cells. mda-7/IL-24 exerts cancer-specific toxicity (apoptosis or toxic autophagy) by promoting ER stress and modulating multiple signal transduction pathways regulating cancer cell growth, invasion, metastasis, survival and angiogenesis. To enhance cancer-selective expression and targeted anti-cancer activity of mda-7/IL-24 we created a tropism-modified Cancer Terminator Virus (Ad.5/3-CTV), which selectively replicates in cancer cells producing robust expression of mda-7/IL-24. We now show that Ad.5/3-CTV induces profound neuroblastoma anti-proliferative activity and apoptosis in a caspase 3/9-independent manner both in vitro and in vivo in a tumor xenograft model. Ad.5/3-CTV promotes these effects through a unique pathway involving apoptosis inducing factor (AIF) translocation into the nucleus. Inhibiting AIF rescued neuroblastoma cells from Ad.5/3-CTV-induced cell death, whereas pan-caspase inhibition failed to promote survival. Ad.5/3-CTV infection of neuroblastoma cells increased ATM phosphorylation instigating nuclear translocation and increased γ–H2AX, triggering nuclear translocation and intensified expression of AIF. These results were validated further using two ATM small molecule inhibitors that attenuated PARP cleavage by inhibiting γ–H2AX, which in turn inhibited AIF changes in Ad.5/3-CTV-infected neuroblastoma cells. Taken together, we elucidate a novel pathway for mda-7/IL-24-induced caspase-independent apoptosis in neuroblastoma cells mediated through modulation of AIF, ATM and γ–H2AX. PMID:27197168

MicroRNA-128b suppresses tumor growth and promotes apoptosis by targeting A2bR in gastric cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ping; Guo, Xueyan; Zong, Wei

2015-11-27

MicroRNAs (miRNAs) play crucial roles in the development and progression of human cancers, including gastric cancer (GC). The discovery of miRNAs may provide a new and powerful tool for studying the mechanism, diagnosis, and treatment of GC. In this study, we aimed to investigate the role and mechanism of miR-128b in the development and progression of GC. Quantitative real-time PCR (qRT-PCR) was used to measure the expression level of miR-128b in GC tissues and cell lines. We found that miR-128b was significantly down-regulated in GC tissues and cell lines. In addition, over-expression of miR-128b inhibited GC cell proliferation, migration andmore » invasion of GC cells in vitro. Gain-of-function in vitro experiments further showed that the miR-128b mimic significantly promoted GC cell apoptosis. Subsequent dual-luciferase reporter assay identified one of the proto-oncogene A2bR as direct target of miR-128b. Therefore, our results indicate that miR-128b is a proto-oncogene miRNA that can suppresses GC proliferation and migration through down-regulation of the oncogene gene A2bR. Taken together, our results indicate that miR-128b could serve as a potential diagnostic biomarker and therapeutic option for human GC in the near future. - Highlights: • The expression of MiR-128b is significantly down-regulated in GC tissues and cell lines. • Ectopic expression of miR-128b directly affects cell proliferation, migration and invasion in vitro. • Overexpression of miR-128b increases apoptosis in GC cells. • A2bR is a candidate target gene of miR-128b. • MiR-128b represses cell proliferation, migration and invasion and promotes apoptosis by targeting A2bR in GC.« less

RUNX3 is oncogenic in natural killer/T-cell lymphoma and is transcriptionally regulated by MYC

PubMed Central

Selvarajan, V; Osato, M; Nah, G S S; Yan, J; Chung, T-H; Voon, D C-C; Ito, Y; Ham, M F; Salto-Tellez, M; Shimizu, N; Choo, S-N; Fan, S; Chng, W-J; Ng, S-B

2017-01-01

RUNX3, runt-domain transcription factor, is a master regulator of gene expression in major developmental pathways. It acts as a tumor suppressor in many cancers but is oncogenic in certain tumors. We observed upregulation of RUNX3 mRNA and protein expression in nasal-type extranodal natural killer (NK)/T-cell lymphoma (NKTL) patient samples and NKTL cell lines compared to normal NK cells. RUNX3 silenced NKTL cells showed increased apoptosis and reduced cell proliferation. Potential binding sites for MYC were identified in the RUNX3 enhancer region. Chromatin immunoprecipitation–quantitative PCR revealed binding activity between MYC and RUNX3. Co-transfection of the MYC expression vector with RUNX3 enhancer reporter plasmid resulted in activation of RUNX3 enhancer indicating that MYC positively regulates RUNX3 transcription in NKTL cell lines. Treatment with a small-molecule MYC inhibitor (JQ1) caused significant downregulation of MYC and RUNX3, leading to apoptosis in NKTL cells. The growth inhibition resulting from depletion of MYC by JQ1 was rescued by ectopic MYC expression. In summary, our study identified RUNX3 overexpression in NKTL with functional oncogenic properties. We further delineate that MYC may be an important upstream driver of RUNX3 upregulation and since MYC is upregulated in NKTL, further study on the employment of MYC inhibition as a therapeutic strategy is warranted. PMID:28119527

Downregulation of miR‑135a predicts poor prognosis in acute myeloid leukemia and regulates leukemia progression via modulating HOXA10 expression.

PubMed

Xu, Hongwei; Wen, Quan

2018-05-23

MicroRNA‑135a (miR‑135a) has been shown to exert important roles in various human cancer types, such as glioblastoma, thyroid carcinoma and renal carcinoma. However, the function of miR‑135a in acute myeloid leukemia (AML) remains largely unknown. In the present study, it was demonstrated that miR‑135a expression was significantly downregulated in AML cells compared with normal control cells. Furthermore, the downregulation of miR‑135a in patients with AML predicted poor prognosis. Through functional experiments, overexpression of miR‑135a was demonstrated to significantly inhibit the proliferation and cell cycle of AML cells, while it promoted cellular apoptosis. miR‑135a directly targeted HOXA10 in AML cells. miR‑135a overexpression significantly suppressed the mRNA and protein levels of HOXA10 in AML cells. Moreover, there was an inverse association between miR‑135a expression and HOXA10 level in AML samples. Additionally, by ectopic expression of HOXA10, restoration of HOXA10 significantly abolished the effects of miR‑135a overexpression on AML cell proliferation, cell cycle and apoptosis. In conclusion, the present study demonstrated that miR‑135a serves as a tumor suppressor in AML by targeting HOXA10, and miR‑135a may be a promising prognostic biomarker for AML patients.

Hsa-miR-875-5p exerts tumor suppressor function through down-regulation of EGFR in colorectal carcinoma (CRC)

PubMed Central

Li, Qi; Xue, Peng; Chen, Zhixiao; Dong, Xiao; Xue, Ying

2016-01-01

Hsa-miRNA-875-5p (miR-875-5p) has recently been discovered to have anticancer efficacy in different organs. However, the role of miR-875-5p on colorectal carcinoma (CRC) is still ambiguous. In this study, we investigated the role of miR-875-5p on the development of CRC. The results indicated that miR-875-5p was significantly down-regulated in primary tumor tissues and very low levels were found in CRC cell lines. Ectopic expression of miR-875-5p in CRC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-875-5p induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-875-5p inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene EGFR was revealed to be a putative target of miR-875-5p, which was inversely correlated with miR-875-5p expression in CRC. Taken together, our results demonstrated that miR-875-5p played a pivotal role on CRC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic EGFR. PMID:27302926

Hsa-miR-875-5p exerts tumor suppressor function through down-regulation of EGFR in colorectal carcinoma (CRC).

PubMed

Zhang, Tiening; Cai, Xun; Li, Qi; Xue, Peng; Chen, Zhixiao; Dong, Xiao; Xue, Ying

2016-07-05

Hsa-miRNA-875-5p (miR-875-5p) has recently been discovered to have anticancer efficacy in different organs. However, the role of miR-875-5p on colorectal carcinoma (CRC) is still ambiguous. In this study, we investigated the role of miR-875-5p on the development of CRC. The results indicated that miR-875-5p was significantly down-regulated in primary tumor tissues and very low levels were found in CRC cell lines. Ectopic expression of miR-875-5p in CRC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-875-5p induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-875-5p inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene EGFR was revealed to be a putative target of miR-875-5p, which was inversely correlated with miR-875-5p expression in CRC. Taken together, our results demonstrated that miR-875-5p played a pivotal role on CRC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic EGFR.

Inhibition of Oncogenic Transcription Factor REL by the Natural Product Derivative Calafianin Monomer 101 Induces Proliferation Arrest and Apoptosis in Human B-Lymphoma Cell Lines.

PubMed

Yeo, Alan T; Chennamadhavuni, Spandan; Whitty, Adrian; Porco, John A; Gilmore, Thomas D

2015-04-23

Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.

RAD18 mediates resistance to ionizing radiation in human glioma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Chen; Wang, Hongwei; Cheng, Hongbin

Highlights: • RAD18 is an important mediator of the IR-induced resistance in glioma cell lines. • RAD18 overexpression confers resistance to IR-mediated apoptosis. • The elevated expression of RAD18 is associated with recurrent GBM who underwent IR therapy. - Abstract: Radioresistance remains a major challenge in the treatment of glioblastoma multiforme (GBM). RAD18 a central regulator of translesion DNA synthesis (TLS), has been shown to play an important role in regulating genomic stability and DNA damage response. In the present study, we investigate the relationship between RAD18 and resistance to ionizing radiation (IR) and examined the expression levels of RAD18more » in primary and recurrent GBM specimens. Our results showed that RAD18 is an important mediator of the IR-induced resistance in GBM. The expression level of RAD18 in glioma cells correlates with their resistance to IR. Ectopic expression of RAD18 in RAD18-low A172 glioma cells confers significant resistance to IR treatment. Conversely, depletion of endogenous RAD18 in RAD18-high glioma cells sensitized these cells to IR treatment. Moreover, RAD18 overexpression confers resistance to IR-mediated apoptosis in RAD18-low A172 glioma cells, whereas cells deficient in RAD18 exhibit increased apoptosis induced by IR. Furthermore, knockdown of RAD18 in RAD18-high glioma cells disrupts HR-mediated repair, resulting in increased accumulation of DSB. In addition, clinical data indicated that RAD18 was significantly higher in recurrent GBM samples that were exposed to IR compared with the corresponding primary GBM samples. Collectively, our findings reveal that RAD18 may serve as a key mediator of the IR response and may function as a potential target for circumventing IR resistance in human GBM.« less

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity[OPEN

PubMed Central

Rebocho, Alexandra B.

2016-01-01

Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth. PMID:27553356

Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements

PubMed Central

Neuman, Sarah D.; Bashirullah, Arash; Kumar, Justin P.

2016-01-01

The eyes absent (eya) gene of the fruit fly, Drosophila melanogaster, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of eya leads to the complete elimination of the compound eye while forced expression of eya in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina eya is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. In this report we explore the mechanisms by which eya expression is spatially and temporally governed in the developing eye. We demonstrate that multiple cis-regulatory elements function cooperatively to control eya transcription and that spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, we show that the loss of eya expression in sine oculis (so) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis. PMID:27930646

Ectopic Expression of Retrotransposon-Derived PEG11/RTL1 Contributes to the Callipyge Muscular Hypertrophy

PubMed Central

Xu, Xuewen; Ectors, Fabien; Davis, Erica E.; Pirottin, Dimitri; Cheng, Huijun; Farnir, Frédéric; Hadfield, Tracy; Cockett, Noelle; Charlier, Carole; Georges, Michel; Takeda, Haruko

2015-01-01

The callipyge phenotype is an ovine muscular hypertrophy characterized by polar overdominance: only heterozygous + Mat /CLPG Pat animals receiving the CLPG mutation from their father express the phenotype. + Mat /CLPG Pat animals are characterized by postnatal, ectopic expression of Delta-like 1 homologue (DLK1) and Paternally expressed gene 11/Retrotransposon-like 1 (PEG11/RTL1) proteins in skeletal muscle. We showed previously in transgenic mice that ectopic expression of DLK1 alone induces a muscular hypertrophy, hence demonstrating a role for DLK1 in determining the callipyge hypertrophy. We herein describe newly generated transgenic mice that ectopically express PEG11 in skeletal muscle, and show that they also exhibit a muscular hypertrophy phenotype. Our data suggest that both DLK1 and PEG11 act together in causing the muscular hypertrophy of callipyge sheep. PMID:26474044

The Lin28/Let-7 System in Early Human Embryonic Tissue and Ectopic Pregnancy

PubMed Central

Steffani, Liliana; Martínez, Sebastián; Monterde, Mercedes; Ferri, Blanca; Núñez, Maria Jose; AinhoaRomero-Espinós; Zamora, Omar; Gurrea, Marta; Sangiao-Alvarellos, Susana; Vega, Olivia; Simón, Carlos; Pellicer, Antonio; Tena-Sempere, Manuel

2014-01-01

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7–9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans. PMID:24498170

Homeostatic regulatory role of Pokemon in NF-κB signaling: stimulating both p65 and IκBα expression in human hepatocellular carcinoma cells.

PubMed

Zhang, Nan-Nan; Sun, Qin-Sheng; Chen, Zhe; Liu, Feng; Jiang, Yu-Yang

2013-01-01

NF-κB consists of p50, p65 (RelA), p52, c-Rel, and RelB, and among them p65 is a representative protein to investigate the regulation and function of this signaling. NF-κB integrates inflammation and carcinogenesis and regulates the expression of a variety of genes in response to immunity, inflammation, and apoptosis. IκBα acts as an inhibitor of NF-κB through forming an inactive NF-κB/IκBα complex. Pokemon is a ubiquitous transcription factor involved in different signaling pathways, playing a pivotal role in cell proliferation, anti-apoptosis, embryonic development, and maintenance. In this study, we found that p65 and IκBα are both novel regulatory targets of Pokemon. Ectopic expression of Pokemon in immortalized liver cells HL7702 enhanced p65 and IκBα expression, whereas silencing of Pokemon in hepatocellular carcinoma cells QGY7703 reduced cellular p65 levels. ChIP assay and targeted mutagenesis revealed that Pokemon directly binds to the element of -434 to -430 bp in p65 promoter and of -453 to -448 bp in IκBα promoter and stimulates luciferase reporter gene expression. Co-transfection of Pokemon with p65 or IκBα promoter-reporter notably enhanced their promoter activity. These data suggest that Pokemon activates the expression of both p65 and IκBα by sequence-specific binding to their promoters and plays a dual role in regulating NF-κB signaling.

Tethered Hsp90 Inhibitors Carrying Optical or Radioiodinated Probes Reveal Selective Internalization of Ectopic Hsp90 in Malignant Breast Tumor Cells

PubMed Central

Barrott, Jared J.; Hughes, Philip F.; Osada, Takuya; Yang, Xiao-Yi; Hartman, Zachary C.; Loiselle, David R.; Spector, Neil L.; Neckers, Len; Rajaram, Narasimhan; Hu, Fangyao; Ramanujam, Nimmi; Vaidyanathan, Ganesan; Zalutsky, Michael R.; Lyerly, H. Kim; Haystead, Timothy A.

2013-01-01

Summary Hsp90 inhibitors have demonstrated unusual selectivity for tumor cells despite its ubiquitous expression. This phenomenon has remained unexplained but could be influenced by ectopically expressed Hsp90 in tumors. We have synthesized novel Hsp90 inhibitors that can carry optical or radioiodinated probes via a PEG tether. We show that these tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein is occurring at the plasma membrane. In mice, we show exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors targeting ectopic Hsp90 can be used to detect breast cancer malignancies through non-invasive imaging. PMID:24035283

F-box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation

PubMed Central

Chen, Bill B.; Glasser, Jennifer R.; Coon, Tiffany A.; Zou, Chunbin; Miller, Hannah L.; Fenton, Moon; McDyer, John F.; Boyiadzis, Michael

2012-01-01

Hematologic maligancies exhibit a growth advantage by up-regulation of components within the molecular apparatus involved in cell-cycle progression. The SCF (Skip-Cullin1-F-box protein) E3 ligase family provides homeostatic feedback control of cell division by mediating ubiquitination and degradation of cell-cycle proteins. By screening several previously undescribed E3 ligase components, we describe the behavior of a relatively new SCF subunit, termed FBXL2, that ubiquitinates and destabilizes cyclin D2 protein leading to G0 phase arrest and apoptosis in leukemic and B-lymphoblastoid cell lines. FBXL2 expression was strongly suppressed, and yet cyclin D2 protein levels were robustly expressed in acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) patient samples. Depletion of endogenous FBXL2 stabilized cyclin D2 levels, whereas ectopically expressed FBXL2 decreased cyclin D2 lifespan. FBXL2 did not bind a phosphodegron within its substrate, which is typical of other F-box proteins, but uniquely targeted a calmodulin-binding signature within cyclin D2 to facilitate its polyubiquitination. Calmodulin competes with the F-box protein for access to this motif where it bound and protected cyclin D2 from FBXL2. Calmodulin reversed FBXL2-induced G0 phase arrest and attenuated FBXL2-induced apoptosis of lymphoblastoid cells. These results suggest an antiproliferative effect of SCFFBXL2 in lymphoproliferative malignancies. PMID:22323446

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion.

PubMed

López, Jesús Adrián; Alvarez-Salas, Luis Marat

2011-06-10

MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology. Copyright © 2011 Elsevier Inc. All rights reserved.

Rotenone Induction of Hydrogen Peroxide Inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E Pathways, Leading to Neuronal Apoptosis

PubMed Central

Zhou, Qian; Liu, Chunxiao; Liu, Wen; Zhang, Hai; Zhang, Ruijie; Liu, Jia; Zhang, Jinfei; Xu, Chong; Liu, Lei; Huang, Shile; Chen, Long

2015-01-01

Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinson’s disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H2O2) involved, which was further verified by imaging intracellular H2O2 using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H2O2-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H2O2 and cell apoptosis. Furthermore, we noticed that rotenone-induced H2O2 was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H2O2 inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants. PMID:25304210

Nrf2 mediates redox adaptation in NOX4-overexpressed non-small cell lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Qipeng; Yao, Bei; Li, Ning

The redox adaptation mechanisms in cancer cells are very complex and remain largely unclear. Our previous studies have confirmed that NADPH oxidase 4 (NOX4) is abundantly expressed in non-small cell lung cancer (NSCLC) and confers apoptosis resistance on NSCLC cells. However, the comprehensive mechanisms for NOX4-mediated oxidative resistance of cancer cells remain still undentified. The present study found that NOX4-derived H{sub 2}O{sub 2} enhanced the nuclear factor erythroid 2-related factor 2 (Nrf2) stability via disruption of redox-dependent proteasomal degradation and stimulated its activity through activation of PI3K signaling. Specifically, the results showed that ectopic NOX4 expression did not induce apoptosismore » of A549 cells; however, inhibition of Nrf2 resulted in obvious apoptotic death of NOX4-overexpressed A549 cells, accompanied by a significant increase in H{sub 2}O{sub 2} level and decrease in GSH content. Besides, inhibition of Nrf2 could suppress cell growth and efficiently reverse the enhancement effect of NOX4 on cell growth. The in vivo data confirmed that inhibition of Nrf2 could interfere apoptosis resistance in NOX4-overexpressed A549 tumors and led to cell growth inhibition. In conclusion, these results reveal that Nrf2 is critically involved in redox adaptation regulation in NOX4-overexpressed NSCLC cells. Therefore, NOX4 and Nrf2 may be promising combination targets against malignant progression of NSCLC. - Highlights: • NOX4-derived H{sub 2}O{sub 2} upregulates Nrf2 expression and activity in NSCLC. • Nrf2 confers apoptosis resistance in NOX4-overexpressed NSCLC cells. • Inhibition of Nrf2 reverses the enhancement effect of NOX4 on cell growth.« less

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells.

PubMed

He, Jinpeng; Feng, Xiu; Hua, Junrui; Wei, Li; Lu, Zhiwei; Wei, Wenjun; Cai, Hui; Wang, Bing; Shi, Wengui; Ding, Nan; Li, He; Zhang, Yanan; Wang, Jufang

2017-10-18

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3'-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer

PubMed Central

Burgess, Joshua T.; Bolderson, Emma; Saunus, Jodi M.; Zhang, Shu-Dong; Reid, Lynne E.; McNicol, Anne Marie; Lakhani, Sunil R.; Cuff, Katharine; Richard, Kerry; Richard, Derek J.; O'Byrne, Kenneth J.

2016-01-01

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation. PMID:27637080

SASH1 mediates sensitivity of breast cancer cells to chloropyramine and is associated with prognosis in breast cancer.

PubMed

Burgess, Joshua T; Bolderson, Emma; Saunus, Jodi M; Zhang, Shu-Dong; Reid, Lynne E; McNicol, Anne Marie; Lakhani, Sunil R; Cuff, Katharine; Richard, Kerry; Richard, Derek J; O'Byrne, Kenneth J

2016-11-08

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation.

Brief Reports: Nfix Promotes Survival of Immature Hematopoietic Cells via Regulation of c-Mpl.

PubMed

Hall, Trent; Walker, Megan; Ganuza, Miguel; Holmfeldt, Per; Bordas, Marie; Kang, Guolian; Bi, Wenjian; Palmer, Lance E; Finkelstein, David; McKinney-Freeman, Shannon

2018-02-12

Hematopoietic stem and progenitor cells (HSPCs) are necessary for life-long blood production and replenishment of the hematopoietic system during stress. We recently reported that nuclear factor I/X (Nfix) promotes HSPC survival post-transplant. Here, we report that ectopic expression of Nfix in primary mouse HSPCs extends their ex vivo culture from about 20 to 40 days. HSPCs overexpressing Nfix display hypersensitivity to supportive cytokines and reduced apoptosis when subjected to cytokine deprivation relative to controls. Ectopic Nfix resulted in elevated levels of c-Mpl transcripts and cell surface protein on primary murine HSPCs as well as increased phosphorylation of STAT5, which is known to be activated down-stream of c-MPL. Blocking c-MPL signaling by removal of thrombopoietin or addition of a c-MPL neutralizing antibody negated the antiapoptotic effect of Nfix overexpression on cultured HSPCs. Furthermore, NFIX was capable of binding to and transcriptionally activating a proximal c-Mpl promoter fragment. In sum, these data suggest that NFIX-mediated upregulation of c-Mpl transcription can protect primitive hematopoietic cells from stress ex vivo. Stem Cells 2018. © AlphaMed Press 2018.

MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai, Shouping; Wang, Xianjun; Li, Xiao

MicroRNA-139-5p was identified to be significantly down-regulated in glioblastoma multiform (GBM) by miRNA array. In this report we aimed to clarify its biological function, molecular mechanisms and direct target gene in GBM. Twelve patients with GBM were analyzed for the expression of miR-139-5p by quantitative RT-PCR. miR-139-5p overexpression was established by transfecting miR-139-5p-mimic into U87MG and T98G cells, and its effects on cell proliferation were studied using MTT assay and colony formation assays. We concluded that ectopic expression of miR-139-5p in GBM cell lines significantly suppressed cell proliferation and inducing apoptosis. Bioinformatics coupled with luciferase and western blot assays alsomore » revealed that miR-139-5p suppresses glioma cell proliferation by targeting ELTD1 and regulating cell cycle. - Highlights: • miR-139-5p is downregulated in GBM. • miR-139-5p regulates cell proliferation through inducing apoptosis. • miR-139-5p regulates glioblastoma tumorigenesis by targeting 3′UTR of ELTD1. • miR-139-5p is involved in cell cycle regulation.« less

E2F1-mediated human POMC expression in ectopic Cushing's syndrome.

PubMed

Araki, Takako; Liu, Ning-Ai; Tone, Yukiko; Cuevas-Ramos, Daniel; Heltsley, Roy; Tone, Masahide; Melmed, Shlomo

2016-11-01

Cushing's syndrome is caused by excessive adrenocorticotropic hormone (ACTH) secretion derived from pituitary corticotroph tumors (Cushing disease) or from non-pituitary tumors (ectopic Cushing's syndrome). Hypercortisolemic features of ectopic Cushing's syndrome are severe, and no definitive treatment for paraneoplastic ACTH excess is available. We aimed to identify subcellular therapeutic targets by elucidating transcriptional regulation of the human ACTH precursor POMC (proopiomelanocortin) and ACTH production in non-pituitary tumor cells and in cell lines derived from patients with ectopic Cushing's syndrome. We show that ectopic hPOMC transcription proceeds independently of pituitary-specific Tpit/Pitx1 and demonstrate a novel E2F1-mediated transcriptional mechanism regulating hPOMC We identify an E2F1 cluster binding to the proximal hPOMC promoter region (-42 to +68), with DNA-binding activity determined by the phosphorylation at Ser-337. hPOMC mRNA expression in cancer cells was upregulated (up to 40-fold) by the co-expression of E2F1 and its heterodimer partner DP1. Direct and indirect inhibitors of E2F1 activity suppressed hPOMC gene expression and ACTH by modifying E2F1 DNA-binding activity in ectopic Cushing's cell lines and primary tumor cells, and also suppressed paraneoplastic ACTH and cortisol levels in xenografted mice. E2F1-mediated hPOMC transcription is a potential target for suppressing ACTH production in ectopic Cushing's syndrome. © 2016 Society for Endocrinology.

Star-PAP, a poly(A) polymerase, functions as a tumor suppressor in an orthotopic human breast cancer model.

PubMed

Yu, C; Gong, Y; Zhou, H; Wang, M; Kong, L; Liu, J; An, T; Zhu, H; Li, Y

2017-02-02

Star-PAP is a noncanonical poly(A) polymerase and required for the expression of a select set of mRNAs. However, the pathological role of Star-PAP in cancer largely remains unknown. In this study, we observed decreased expression of Star-PAP in breast cancer cell lines and tissues. Ectopic Star-PAP expression inhibited proliferation as well as colony-forming ability of breast cancer cells. In breast cancer patients, high levels of Star-PAP correlated with an improved prognosis. Moreover, by regulating the expression of BIK (BCL2-interacting killer), Star-PAP induced apoptosis of breast cancer cells through the mitochondrial pathway. The growth of breast cancer xenografts in NOD/SCID mice was also inhibited by the doxycycline-induced Star-PAP overexpression. Furthermore, Star-PAP sensitized breast cancer cells to chemotherapy drugs both in vitro and in vivo. In mammary epithelial cells, Star-PAP knockdown partially transformed these cells and induced them to undergo epithelial-mesenchymal transition (EMT). These findings suggested that Star-PAP possesses tumor-suppressing activity and can be a valuable target for developing new cancer therapeutic strategies.

Developmental Role and Auxin Responsiveness of Class III Homeodomain Leucine Zipper Gene Family Members in Rice1[C][W][OA

PubMed Central

Itoh, Jun-Ichi; Hibara, Ken-Ichiro; Sato, Yutaka; Nagato, Yasuo

2008-01-01

Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner. PMID:18567825

Methuosis

PubMed Central

Maltese, William A.; Overmeyer, Jean H.

2015-01-01

Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death. PMID:24726643

Active Depletion of Host Cell Inhibitor-of-Apoptosis Proteins Triggers Apoptosis upon Baculovirus DNA Replication▿

PubMed Central

Vandergaast, Rianna; Schultz, Kimberly L. W.; Cerio, Rebecca J.; Friesen, Paul D.

2011-01-01

Apoptosis is an important antivirus defense by virtue of its impact on virus multiplication and pathogenesis. To define molecular mechanisms by which viruses are detected and the apoptotic response is initiated, we examined the antiviral role of host inhibitor-of-apoptosis (IAP) proteins in insect cells. We report here that the principal IAPs, DIAP1 and SfIAP, of the model insects Drosophila melanogaster and Spodoptera frugiperda, respectively, are rapidly depleted and thereby inactivated upon infection with the apoptosis-inducing baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Virus-induced loss of these host IAPs triggered caspase activation and apoptotic death. Elevation of IAP levels by ectopic expression repressed caspase activation. Loss of host IAP in both species was triggered by AcMNPV DNA replication. By using selected inhibitors, we found that virus-induced IAP depletion was mediated in part by the proteasome but not by caspase cleavage. Consistent with this conclusion, mutagenic disruption of the SfIAP RING motif, which acts as an E3 ubiquitin ligase, stabilized SfIAP during infection. Importantly, SfIAP was also stabilized upon the removal of its 99-residue N-terminal leader, which serves as a critical determinant of IAP turnover. These data indicated that a host pathway initiated by virus DNA replication and acting through instability motifs embedded within IAP triggers IAP depletion and thereby causes apoptosis. Taken together, the results of our study suggest that host modulation of cellular IAP levels is a conserved mechanism by which insects mount an apoptotic antiviral response. Thus, host IAPs may function as critical sentinels of virus invasion in insects. PMID:21653668

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

PubMed

Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

2009-08-07

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

Transcription factor Sox4 is required for PUMA-mediated apoptosis induced by histone deacetylase inhibitor, TSA.

PubMed

Jang, Sang-Min; Kang, Eun-Jin; Kim, Jung-Woong; Kim, Chul-Hong; An, Joo-Hee; Choi, Kyung-Hee

2013-08-23

PUMA is a crucial regulator of apoptotic cell death mediated by p53-dependent and p53-independent mechanisms. In many cancer cells, PUMA expression is induced in response to DNA-damaging reagent in a p53-dependent manner. However, few studies have investigated transcription factors that lead to the induction of PUMA expression via p53-independent apoptotic signaling. In this study, we found that the transcription factor Sox4 increased PUMA expression in response to trichostatin A (TSA), a histone deacetylase inhibitor in the p53-null human lung cancer cell line H1299. Ectopic expression of Sox4 led to the induction of PUMA expression at the mRNA and protein levels, and TSA-mediated up-regulation of PUMA transcription was repressed by the knockdown of Sox4. Using luciferase assays and chromatin immunoprecipitation, we also determined that Sox4 recruits p300 on the PUMA promoter region and increases PUMA gene expression in response to TSA treatment. Taken together, these results suggest that Sox4 is required for p53-independent apoptotic cell death mediated by PUMA induction via TSA treatment. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

IL-12p40 impairs mesenchymal stem cell-mediated bone regeneration via CD4+ T cells

PubMed Central

Xu, Jiajia; Wang, Yiyun; Li, Jing; Zhang, Xudong; Geng, Yiyun; Huang, Yan; Dai, Kerong; Zhang, Xiaoling

2016-01-01

Severe or prolonged inflammatory response caused by infection or biomaterials leads to delayed healing or bone repair failure. This study investigated the important roles of the proinflammatory cytokines of the interleukin-12 (IL-12) family, namely, IL-12 and IL-23, in the inflammation-mediated inhibition of bone formation in vivo. IL-12p40−/− mice lacking IL-12 and IL-23 exhibited enhanced bone formation. IL-12 and IL-23 indirectly inhibited bone marrow mesenchymal stem cell (BMMSC) differentiation by stimulating CD4+ T cells to increase interferon γ (IFN-γ) and IL-17 levels. Mechanistically, IL-17 synergistically enhanced IFN-γ-induced BMMSC apoptosis. Moreover, INF-γ and IL-17 exerted proapoptotic effects by upregulating the expression levels of Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as by activating the caspase cascade in BMMSCs. IL-12p40 depletion in mice could promote ectopic bone formation. Thus, IL-12p40 is an attractive therapeutic target to overcome the inflammation-mediated inhibition of bone formation in vivo. PMID:27472064

Thymic B Cell-Mediated Attack of Thymic Stroma Precedes Type 1 Diabetes Development

PubMed Central

Pinto, Ana Isabel; Smith, Jennifer; Kissack, Miriam R.; Hogg, Karen G.; Green, E. Allison

2018-01-01

Type 1 diabetes (T1D) results from a coordinated autoimmune attack of insulin producing beta cells in the pancreas by the innate and adaptive immune systems, beta cell death being predominantly T cell-mediated. In addition to T cells, peripheral B cells are important in T1D progression. The thymus of mice and man also contains B cells, and lately they have been linked to central tolerance of T cells. The role of thymic B cells in T1D is undefined. Here, we show there are abnormalities in the thymic B cell compartment before beta cell destruction and T1D manifestation. Using non-obese diabetic (NOD) mice, we document that preceding T1D development, there is significant accumulation of thymic B cells-partly through in situ development- and the putative formation of ectopic germinal centers. In addition, in NOD mice we quantify thymic plasma cells and observe in situ binding of immunoglobulins to undefined antigens on a proportion of medullary thymic epithelial cells (mTECs). By contrast, no ectopic germinal centers or pronounced intrathymic autoantibodies are detectable in animals not genetically predisposed to developing T1D. Binding of autoantibodies to thymic stroma correlates with apoptosis of mTECs, including insulin-expressing cells. By contrast, apoptosis of mTECs was decreased by 50% in B cell-deficient NOD mice suggesting intrathymic autoantibodies may selectively target certain mTECs for destruction. Furthermore, we observe that these thymic B cell-associated events correlated with an increased prevalence of premature thymic emigration of T cells. Together, our data suggest that the thymus may be a principal autoimmune target in T1D and contributes to disease progression.

Endometrial cysteine-rich secretory protein 3 is inhibited by human chorionic gonadotrophin, and is increased in the decidua of tubal ectopic pregnancy.

PubMed

Horne, A W; Duncan, W C; King, A E; Burgess, S; Lourenco, P C; Cornes, P; Ghazal, P; Williams, A R; Udby, L; Critchley, H O D

2009-05-01

Ectopic pregnancy (EP) remains a considerable cause of morbidity and occasional mortality. Currently, there is no reliable test to differentiate ectopic from intrauterine gestation. We have previously used array technology to demonstrate that differences in gene expression in decidualized endometrium from women with ectopic and intrauterine gestations could be used to identify candidate diagnostic biomarkers for EP. The aim of this study was to further investigate the decidual gene with the highest fold increase in EP, cysteine-rich secretory protein-3 (CRISP-3). Decidualized endometrium from gestation-matched women undergoing surgical termination of pregnancy (n = 8), evacuation of uterus for miscarriage (n = 6) and surgery for EP (n = 11) was subjected to quantitative RT-PCR, morphological assessment, immunohistochemistry and western blot analysis. Sera were analysed for progesterone and human chorionic gonadotrophin (hCG) levels. Immortalized endometrial epithelial cells were cultured with physiological concentrations of hCG. CRISP-3 mRNA and protein expression were greater in endometrium from ectopic when compared with intrauterine pregnancies (P < 0.05). CRISP-3 protein was localized to epithelium and granulocytes of endometrium. CRISP-3 serum concentrations were not different in women with ectopic compared with intrauterine pregnancies. CRISP-3 expression in endometrium was not related to the degree of decidualization or to serum progesterone levels. Endometrial CRISP-3 expression was inversely proportional to serum hCG concentrations (P < 0.001). Stimulation of endometrial epithelial cells with hCG in vitro caused a reduction in CRISP-3 expression (P < 0.01). The measurement of CRISP-3 in endometrium could provide an additional tool in the diagnosis of failing early pregnancy of unknown location. The absence of a local reduction in expression of CRISP-3 in decidualized endometrium of women with EP may be due to reduced exposure to hCG due to the ectopic location of the trophoblast.

LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

PubMed Central

Liu, Xiuju; Huang, Henry; Wilkinson, Scott C.; Zhong, Diansheng; Khuri, Fadlo R.; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

2016-01-01

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells. PMID:26506235

LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions.

PubMed

Liu, Fakeng; Jin, Rui; Liu, Xiuju; Huang, Henry; Wilkinson, Scott C; Zhong, Diansheng; Khuri, Fadlo R; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

2016-01-19

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.

Mesenchyme-specific knockout of ESET histone methyltransferase causes ectopic hypertrophy and terminal differentiation of articular chondrocytes.

PubMed

Lawson, Kevin A; Teteak, Colin J; Zou, Junhui; Hacquebord, Jacques; Ghatan, Andrew; Zielinska-Kwiatkowska, Anna; Fernandes, Russell J; Chansky, Howard A; Yang, Liu

2013-11-08

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.

Ectopic expression of Cripto-1 in transgenic mouse embryos causes hemorrhages, fatal cardiac defects and embryonic lethality

PubMed Central

Lin, Xiaolin; Zhao, Wentao; Jia, Junshuang; Lin, Taoyan; Xiao, Gaofang; Wang, Shengchun; Lin, Xia; Liu, Yu; Chen, Li; Qin, Yujuan; Li, Jing; Zhang, Tingting; Hao, Weichao; Chen, Bangzhu; Xie, Raoying; Cheng, Yushuang; Xu, Kang; Yao, Kaitai; Huang, Wenhua; Xiao, Dong; Sun, Yan

2016-01-01

Targeted disruption of Cripto-1 in mice caused embryonic lethality at E7.5, whereas we unexpectedly found that ectopic Cripto-1 expression in mouse embryos also led to embryonic lethality, which prompted us to characterize the causes and mechanisms underlying embryonic death due to ectopic Cripto-1 expression. RCLG/EIIa-Cre embryos displayed complex phenotypes between embryonic day 14.5 (E14.5) and E17.5, including fatal hemorrhages (E14.5-E15.5), embryo resorption (E14.5-E17.5), pale body surface (E14.5-E16.5) and no abnormal appearance (E14.5-E16.5). Macroscopic and histological examination revealed that ectopic expression of Cripto-1 transgene in RCLG/EIIa-Cre embryos resulted in lethal cardiac defects, as evidenced by cardiac malformations, myocardial thinning, failed assembly of striated myofibrils and lack of heartbeat. In addition, Cripto-1 transgene activation beginning after E8.5 also caused the aforementioned lethal cardiac defects in mouse embryos. Furthermore, ectopic Cripto-1 expression in embryonic hearts reduced the expression of cardiac transcription factors, which is at least partially responsible for the aforementioned lethal cardiac defects. Our results suggest that hemorrhages and cardiac abnormalities are two important lethal factors in Cripto-1 transgenic mice. Taken together, these findings are the first to demonstrate that sustained Cripto-1 transgene expression after E11.5 causes fatal hemorrhages and lethal cardiac defects, leading to embryonic death at E14.5-17.5. PMID:27687577

ADAR1-mediated 3' UTR editing and expression control of antiapoptosis genes fine-tunes cellular apoptosis response.

PubMed

Yang, Chang-Ching; Chen, Yi-Tung; Chang, Yi-Feng; Liu, Hsuan; Kuo, Yu-Ping; Shih, Chieh-Tien; Liao, Wei-Chao; Chen, Hui-Wen; Tsai, Wen-Sy; Tan, Bertrand Chin-Ming

2017-05-25

Adenosine-to-inosine RNA editing constitutes a crucial component of the cellular transcriptome and critically underpins organism survival and development. While recent high-throughput approaches have provided comprehensive documentation of the RNA editome, its functional output remains mostly unresolved, particularly for events in the non-coding regions. Gene ontology analysis of the known RNA editing targets unveiled a preponderance of genes related to apoptosis regulation, among which proto-oncogenes XIAP and MDM2 encode two the most abundantly edited transcripts. To further decode this potential functional connection, here we showed that the main RNA editor ADAR1 directly targets this 3' UTR editing of XIAP and MDM2, and further exerts a negative regulation on the expression of their protein products. This post-transcriptional silencing role was mediated via the inverted Alu elements in the 3' UTR but independent of alteration in transcript stability or miRNA targeting. Rather, we discovered that ADAR1 competes transcript occupancy with the RNA shuttling factor STAU1 to facilitate nuclear retention of the XIAP and MDM2 mRNAs. As a consequence, ADAR1 may acquire functionality in part by conferring spatial distribution and translation efficiency of the target transcripts. Finally, abrogation of ADAR1 expression or catalytic activity elicited a XIAP-dependent suppression of apoptotic response, whereas ectopic expression reversed this protective effect on cell death. Together, our results extended the known functions of ADAR1 and RNA editing to the critical fine-tuning of the intracellular apoptotic signaling and also provided mechanistic explanation for ADAR1's roles in development and tumorigenesis.

Overexpression of E2F3 promotes proliferation of functional human β cells without induction of apoptosis

PubMed Central

Rady, Brian; Chen, Yanmei; Vaca, Pilar; Wang, Qian; Wang, Yong; Salmon, Patrick; Oberholzer, José

2013-01-01

The mechanisms that control proliferation, or lack thereof, in adult human β cells are poorly understood. Controlled induction of proliferation could dramatically expand the clinical application of islet cell transplantation and represents an important component of regenerative approaches to a functional cure of diabetes. Adult human β cells are particularly resistant to common proliferative targets and often dedifferentiate during proliferation. Here we show that expression of the transcription factor E2F3 has a role in regulating β-cell quiescence and proliferation. We found human islets have virtually no expression of the pro-proliferative G1/S transcription factors E2F1–3, but an abundance of inhibitory E2Fs 4–6. In proliferative human insulinomas, inhibitory E2Fs were absent, while E2F3 is expressed. Using this pattern as a “roadmap” for proliferation, we demonstrated that ectopic expression of nuclear E2F3 induced significant expansion of insulin-positive cells in both rat and human islets. These cells did not undergo apoptosis and retained their glucose-responsive insulin secretion, showing the ability to reverse diabetes in mice. Our results suggest that E2F4–6 may help maintain quiescence in human β cells and identify E2F3 as a novel target to induce proliferation of functional β cells. Refinement of this approach may increase the islets available for cell-based therapies and research and could provide important cues for understanding in vivo proliferation of β cells. PMID:23907129

Development of a Model for Chemotherapy-Induced Alopecia: Profiling of Histological Changes in Human Hair Follicles after Chemotherapy.

PubMed

Yoon, Ji-Seon; Choi, Mira; Shin, Chang Yup; Paik, Seung Hwan; Kim, Kyu Han; Kwon, Ohsang

2016-03-01

Optimized research models are required to further understand the pathogenesis and prophylaxis of chemotherapy-induced alopecia. Our aim was to develop a mouse model for chemotherapy-induced alopecia by follicular unit transplantation of human hair follicles onto immunodeficient mice. Twenty-two weeks after transplantation, a single dose of cyclophosphamide (Cph) was administered to mice in the Cph100 (100 mg/kg) and Cph150 (150 mg/kg) groups. On day 6, hair follicles showed dystrophic changes, with swollen dermal papilla and ectopic melanin clumping in the hair bulb. In addition, upregulated expression of apoptotic regulators [P53, Fas/Fas-ligand, tumor necrosis factor-related apoptosis-inducing ligand/tumor necrosis factor-related apoptosis-inducing ligand receptor (TRAIL/TRAIL receptor), and Bax], increased apoptotic matrix keratinocytes, downregulated Ki67 expression, and decreased melanogenic protein in the hair bulb were noted in both groups. After 12 treatment days, hair follicles in Cph100 mice appeared to diminish dystrophic changes. In contrast, hair follicles of Cph150 mice prematurely entered a dystrophic catagen phase after 9 treatment days, and immunofluorescence staining for Ki67 and melanogenic protein expressions was barely visible. Two hair follicle damage response pathways were observed in this model, namely dystrophic anagen (Cph100) and catagen (Cph150) pathways. Our model might be useful for further understanding the impact of chemotherapy on human hair follicles. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation.

PubMed

Costa, Fernanda C; Saito, Angela; Gonçalves, Kaliandra A; Vidigal, Pedro M; Meirelles, Gabriela V; Bressan, Gustavo C; Kobarg, Jörg

2014-12-01

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events. Copyright © 2014 Elsevier B.V. All rights reserved.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade.

PubMed

Caldeira, Joana; Figueiredo, Joana; Brás-Pereira, Catarina; Carneiro, Patrícia; Moreira, Ana M; Pinto, Marta T; Relvas, João B; Carneiro, Fátima; Barbosa, Mário; Casares, Fernando; Janody, Florence; Seruca, Raquel

2015-10-15

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade

PubMed Central

Caldeira, Joana; Figueiredo, Joana; Brás-Pereira, Catarina; Carneiro, Patrícia; Moreira, Ana M.; Pinto, Marta T.; Relvas, João B.; Carneiro, Fátima; Barbosa, Mário; Casares, Fernando; Janody, Florence; Seruca, Raquel

2015-01-01

Epithelial-cadherin (Ecad) deregulation affects cell–cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell–matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression. PMID:26246502

Long noncoding RNA GAS5 suppresses triple negative breast cancer progression through inhibition of proliferation and invasion by competitively binding miR-196a-5p.

PubMed

Li, Shuqin; Zhou, Jun; Wang, Zhaoxin; Wang, Peishun; Gao, Xitao; Wang, Yan

2018-05-21

Triple-negative breast cancer (TNBC) is considered to be the most aggressive and lethal type of breast cancer. Many studies have suggested that the dysfunction of long noncoding RNAs (lncRNAs) is correlated with breast cancer metastasis and progression. Here, we show that levels of the lncRNA, growth arrest-specific transcript 5 (GAS5), are decreased in TNBC tissues, and this down-regulation of GAS5 is associated with an aggressive tumor phenotype in patients, affecting clinical stage, lymph node metastasis and overall survival. Using an ectopic overexpression system in TNBC cells, we found that up-regulation of GAS5 can significantly attenuate proliferation and enhance apoptosis in TNBC cells. Through bioinformatics analysis and verification with qRT-PCR and luciferase assay, we found that GAS5 can bind to miR-196a-5p and there is a negative relationship between GAS5 and miR-196a-5p expression among TNBC patient samples. Furthermore, we demonstrated that overexpression of GAS5 can partially undermine the tumor promotion effect induced by ectopic expression of miR-196a-5p, including invasion and downstream FOXO1/PI3K/AKT signal pathway activation. In our study, GAS5 functioned as a competing endogenous RNA (ceRNA) antagonizing tumor promotion of miR-196a-5p-expressing TNBC cells. These data suggest that GAS5 can suppress TNBC progression by competitively binding miR-196a-5p, therefore GAS5 may be a prognostic biomarker of TNBC. Copyright © 2018. Published by Elsevier Masson SAS.

Expression of a repressor form of the Arabidopsis thaliana transcription factor TCP16 induces the formation of ectopic meristems.

PubMed

Uberti-Manassero, Nora G; Coscueta, Ezequiel R; Gonzalez, Daniel H

2016-11-01

Plants that express a fusion of the Arabidopsis thaliana class I TCP transcription factor TCP16 to the EAR repressor domain develop several phenotypic alterations, including rounder leaves, short petioles and pedicels, and delayed elongation of sepals, petals and anthers. In addition, these plants develop lobed cotyledons and ectopic meristems. Ectopic meristems are formed on the adaxial side of cotyledon petioles and arise from a cleft that is formed at this site. Analysis of the expression of reporter genes indicated that meristem genes are reactivated at the site of emergence of ectopic meristems, located near the bifurcation of cotyledon veins. The plants also show increased transcript levels of the boundary-specific CUP-SHAPED COTYLEDON (CUC) genes. The results suggest that TCP16 is able to modulate the induction of meristematic programs and the differentiation state of plant cells. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Phospholipase C/protein kinase C pathway mediates angiotensin II-dependent apoptosis in neonatal rat cardiac fibroblasts expressing AT1 receptor.

PubMed

Vivar, Raul; Soto, Cristian; Copaja, Miguel; Mateluna, Francisca; Aranguiz, Pablo; Muñoz, Juan Pablo; Chiong, Mario; Garcia, Lorena; Letelier, Alan; Thomas, Walter G; Lavandero, Sergio; Díaz-Araya, Guillermo

2008-08-01

Cardiac fibroblasts are the major non-myocyte cell constituent in the myocardium, and they are involved in heart remodeling. Angiotensin II type 1 receptor (AT1R) mediates the established actions of angiotensin II (Ang II), and changes in its expression have been reported in cardiac fibroblasts after myocardial infarction. However, the AT1R-dependent signaling pathways involved in cardiac fibroblast death remain unknown. Using adenovirus, we ectopically expressed AT1R in cultured neonatal rat cardiac fibroblasts and investigated the role of the phospholipase (PLC)/protein kinase C (PKC) pathway on Ang II-dependent death. Ang II induced cardiac fibroblast death characterized by an early loss of mitochondrial membrane potential, increased Bax/Bcl-2 ratio, caspase-3 activation, and DNA fragmentation. All these effects were prevented by the AT1R antagonist losartan, PLC inhibitor U73122, and PKC inhibitor Gö6976. We conclude that Ang II stimulates the intrinsic apoptotic pathway in cultured cardiac fibroblasts by the AT1R/PLC/PKC signaling pathway.

NR4A3 Suppresses Lymphomagenesis through Induction of Proapoptotic Genes.

PubMed

Deutsch, Alexander J A; Rinner, Beate; Pichler, Martin; Prochazka, Katharina; Pansy, Katrin; Bischof, Marco; Fechter, Karoline; Hatzl, Stefan; Feichtinger, Julia; Wenzl, Kerstin; Frisch, Marie-Therese; Stiegelbauer, Verena; Prokesch, Andreas; Krogsdam, Anne; Sill, Heinz; Thallinger, Gerhard G; Greinix, Hildegard T; Wang, Chenguang; Beham-Schmid, Christine; Neumeister, Peter

2017-05-01

Nuclear orphan receptor NR4A1 exerts an essential tumor suppressor function in aggressive lymphomas. In this study, we investigated the hypothesized contribution of the related NR4A family member NR4A3 to lymphomagenesis. In aggressive lymphoma patients, low expression of NR4A3 was associated with poor survival. Ectopic expression or pharmacological activation of NR4A3 in lymphoma cell lines led to a significantly higher proportion of apoptotic cells. In a mouse NSG xenograft model of lymphoma (stably transduced SuDHL4 cells), NR4A3 expression abrogated tumor growth, compared with vector control and uninduced cells that formed massive tumors. Transcript analysis of four different aggressive lymphoma cell lines overexpressing either NR4A3 or NR4A1 revealed that apoptosis was driven similarly by induction of BAK, Puma, BIK, BIM, BID, and Trail. Overall, our results showed that NR4A3 possesses robust tumor suppressor functions of similar impact to NR4A1 in aggressive lymphomas. Cancer Res; 77(9); 2375-86. ©2017 AACR . ©2017 American Association for Cancer Research.

Navigator-3, a modulator of cell migration, may act as a suppressor of breast cancer progression

PubMed Central

Cohen-Dvashi, Hadas; Ben-Chetrit, Nir; Russell, Roslin; Carvalho, Silvia; Lauriola, Mattia; Nisani, Sophia; Mancini, Maicol; Nataraj, Nishanth; Kedmi, Merav; Roth, Lee; Köstler, Wolfgang; Zeisel, Amit; Yitzhaky, Assif; Zylberg, Jacques; Tarcic, Gabi; Eilam, Raya; Wigelman, Yoav; Will, Rainer; Lavi, Sara; Porat, Ziv; Wiemann, Stefan; Ricardo, Sara; Schmitt, Fernando; Caldas, Carlos; Yarden, Yosef

2015-01-01

Dissemination of primary tumor cells depends on migratory and invasive attributes. Here, we identify Navigator-3 (NAV3), a gene frequently mutated or deleted in human tumors, as a regulator of epithelial migration and invasion. Following induction by growth factors, NAV3 localizes to the plus ends of microtubules and enhances their polarized growth. Accordingly, NAV3 depletion trimmed microtubule growth, prolonged growth factor signaling, prevented apoptosis and enhanced random cell migration. Mathematical modeling suggested that NAV3-depleted cells acquire an advantage in terms of the way they explore their environment. In animal models, silencing NAV3 increased metastasis, whereas ectopic expression of the wild-type form, unlike expression of two, relatively unstable oncogenic mutants from human tumors, inhibited metastasis. Congruently, analyses of > 2,500 breast and lung cancer patients associated low NAV3 with shorter survival. We propose that NAV3 inhibits breast cancer progression by regulating microtubule dynamics, biasing directionally persistent rather than random migration, and inhibiting locomotion of initiated cells. PMID:25678558

MicroRNA-187-3p mitigates non-small cell lung cancer (NSCLC) development through down-regulation of BCL6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Chengcao; Li, Shujun; Wuhan Hospital for the Prevention and Treatment of Occupational Diseases, 430071 Wuhan

2016-02-26

Hsa-microRNA-187-3p (miR-187-3p) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-187-3p on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-187-3p on the development of NSCLC. The results indicated that miR-187-3p was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-187-3p in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay and colony formation assay, through inhibition of BCL6. In addition, miR-187-3p induced apoptosis, as indicated by concomitantly with up-regulation ofmore » the activities of caspase-3 and caspase-7, and inhibited cellular migration and invasiveness through inhibition of BCL6. Further, oncogene BCL6 was revealed to be a putative target of miR-187-3p, which was inversely correlated with miR-187-3p expression in NSCLC. Taken together, our results demonstrated that miR-187-3p played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic BCL6.« less

Short Vegetative Phase-Like MADS-Box Genes Inhibit Floral Meristem Identity in Barley1[W][OA

PubMed Central

Trevaskis, Ben; Tadege, Million; Hemming, Megan N.; Peacock, W. James; Dennis, Elizabeth S.; Sheldon, Candice

2007-01-01

Analysis of the functions of Short Vegetative Phase (SVP)-like MADS-box genes in barley (Hordeum vulgare) indicated a role in determining meristem identity. Three SVP-like genes are expressed in vegetative tissues of barley: Barley MADS1 (BM1), BM10, and Vegetative to Reproductive Transition gene 2. These genes are induced by cold but are repressed during floral development. Ectopic expression of BM1 inhibited spike development and caused floral reversion in barley, with florets at the base of the spike replaced by tillers. Head emergence was delayed in plants that ectopically express BM1, primarily by delayed development after the floral transition, but expression levels of the barley VRN1 gene (HvVRN1) were not affected. Ectopic expression of BM10 inhibited spike development and caused partial floral reversion, where florets at the base of the spike were replaced by inflorescence-like structures, but did not affect heading date. Floral reversion occurred more frequently when BM1 and BM10 ectopic expression lines were grown in short-day conditions. BM1 and BM10 also inhibited floral development and caused floral reversion when expressed in Arabidopsis (Arabidopsis thaliana). We conclude that SVP-like genes function to suppress floral meristem identity in winter cereals. PMID:17114273

[Expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in human and nude mouse ectopic endometrium and the effect of estrogen and progestin on their expression].

PubMed

Lou, Yan-hui; Guo, Xin-hua; Jiang, Hua; Xia, Yu-fang

2010-04-01

To explore the roles of matrix metalloproteinase-1(MMP-1) and tissue inhibitor of metalloproteinase-1(TIMP-1) in the pathogenesis of endometriosis and the effects of estrogen and progestin on their expression. Immunohistochemistry and RT-PCR were employed to detect the expression of MMP-1 and TIMP-1 in the ectopic tissues of 35 patients with endometriosis, 22 eutopic endometrium tissues from women with endometriosis and 28 normal controls. Fifty-nine nude mice were injected with human late secretory endometrial chippings and randomized into estrogen group, progestin group, estrogen-progestin group and control group with corresponding treatments. The implantation rates and graft morphology were observed and MMP-1 and TIMP-1 expressions in the grafts detected by immunohistochemistry. Typical endometrial glands and stroma were observed in all the groups with comparable implantation rates. The administration of progestin was associated with multiple peritoneal implantation sites and significantly larger implants. The transplanted endometria showed proliferative or secretory changes with estrogen or progestin administration. MMP-1 expression significantly increased and TIMP-1 expression decreased with increased MMP-1/TIMP-1 ratio in human and nude mouse ectopic endometria in comparison with those in normal endometria (P<0.05, P<0.01). MMP-1 expression was higher in estrogen and estrogen-progestin groups than in the control group, and was lower in the 3 sexual hormone-treated groups than in the control group. MMP-1 mRNA expression in the eutopic endometrium was significantly higher than that in the normal endometria. Progestrin can not inhibit MMP-1 expression or the effect of estrogen on ectopic endometrium known as progestin resistance. The high expression of MMP-1 and low expression of TIMP-1 in endometriotic tissues confer strong invasiveness of ectopic endometrial tissue, especially in eutopic endometrial tissue, and may play an important role in the pathogenesis of endometriosis.

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Jesus Adrian; Alvarez-Salas, Luis Marat, E-mail: lalvarez@cinvestav.mx

Highlights: {yields} In this study we examine miR-34c-3p and miR-34c-5p functions in SiHa cells. {yields} We study miRNA effect on cell proliferation, anchorage independent growth, apoptosis, cell motility and invasion. {yields} We find that miR-34c-3p and miR-34c-5p inhibition of proliferation and anchorage independent growth are exclusive to SiHa cells. {yields} miR-34c-3p induces apoptosis and inhibits cell motility and invasion in SiHa cells. {yields} In this study we conclude that miR-34c-3p functions as a tumor suppressor differ from miR-34c-5p. -- Abstract: MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effectormore » of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology.« less

Aromatase expression increases the survival and malignancy of estrogen receptor positive breast cancer cells.

PubMed

Mukhopadhyay, Keya De; Liu, Zhao; Bandyopadhyay, Abhik; Kirma, Nameer B; Tekmal, Rajeshwar R; Wang, Shui; Sun, Lu-Zhe

2015-01-01

In postmenopausal women, local estrogen produced by adipose stromal cells in the breast is believed to support estrogen receptor alpha (ERα) positive breast cancer cell survival and growth. This raises the question of how the ERα positive metastatic breast cancer cells survive after they enter blood and lymph circulation, where estrogen level is very low in postmenopausal women. In this study, we show that the aromatase expression increased when ERα positive breast cancer cells were cultured in suspension. Furthermore, treatment with the aromatase substrate, testosterone, inhibited suspension culture-induced apoptosis whereas an aromatase inhibitor attenuated the effect of testosterone suggesting that suspended circulating ERα positive breast cancer cells may up-regulate intracrine estrogen activity for survival. Consistent with this notion, a moderate level of ectopic aromatase expression rendered a non-tumorigenic ERα positive breast cancer cell line not only tumorigenic but also metastatic in female nude mice without exogenous estrogen supplementation. The increased malignant phenotype was confirmed to be due to aromatase expression as the growth of orthotopic tumors regressed with systemic administration of an aromatase inhibitor. Thus, our study provides experimental evidence that aromatase plays an important role in the survival of metastatic ERα breast cancer cells by suppressing anoikis.

Star-PAP, a poly(A) polymerase, functions as a tumor suppressor in an orthotopic human breast cancer model

PubMed Central

Yu, C; Gong, Y; Zhou, H; Wang, M; Kong, L; Liu, J; An, T; Zhu, H; Li, Y

2017-01-01

Star-PAP is a noncanonical poly(A) polymerase and required for the expression of a select set of mRNAs. However, the pathological role of Star-PAP in cancer largely remains unknown. In this study, we observed decreased expression of Star-PAP in breast cancer cell lines and tissues. Ectopic Star-PAP expression inhibited proliferation as well as colony-forming ability of breast cancer cells. In breast cancer patients, high levels of Star-PAP correlated with an improved prognosis. Moreover, by regulating the expression of BIK (BCL2-interacting killer), Star-PAP induced apoptosis of breast cancer cells through the mitochondrial pathway. The growth of breast cancer xenografts in NOD/SCID mice was also inhibited by the doxycycline-induced Star-PAP overexpression. Furthermore, Star-PAP sensitized breast cancer cells to chemotherapy drugs both in vitro and in vivo. In mammary epithelial cells, Star-PAP knockdown partially transformed these cells and induced them to undergo epithelial–mesenchymal transition (EMT). These findings suggested that Star-PAP possesses tumor-suppressing activity and can be a valuable target for developing new cancer therapeutic strategies. PMID:28151486

TGFβ signaling supports survival and metastasis of endometrial cancer cells

PubMed Central

Lei, XiuFen; Wang, Long; Yang, Junhua; Sun, Lu-Zhe

2009-01-01

The association of mutation of the transforming growth factor beta (TGFβ) type II receptor (RII) with microsatellite instability revealed a significant molecular mechanism of tumorigenesis and tumor progression in gastrointestinal carcinomas with DNA replication error. However, mutation of RII is rare in other types of carcinomas with microsatellite instability including endometrial adenocarcinoma suggesting that TGFβ receptor signaling may be necessary for tumor progression. To test this hypothesis, we abrogated TGFβ signaling with ectopic expression of a dominant-negative RII (DNRII) in human endometrial carcinoma HEC-1-A cells with microsatellite instability. Our study showed that over-expression of DNRII blocked the TGFβ signaling, inhibited anchorage-dependent and -independent growth, and stimulated apoptosis in vitro. Interestingly, the expression of DNRII expression showed little effect on tumor growth of subcutaneously inoculated cells in vivo. On the other hand, the DNRII cells showed more epithelial features whereas the control cells showed more mesenchymal features suggesting a reversal of autocrine TGFβ-induced epithelial–mesenchymal transition (EMT). Consistent with these findings, DNRII cells were much less migratory and invasive in vitro and metastatic in vivo than the control cells. Therefore, an intact TGFβ signaling pathway appears necessary for the metastatic phenotypes of this carcinoma model. PMID:20622970

Role of iron overload-induced macrophage apoptosis in the pathogenesis of peritoneal endometriosis.

PubMed

Pirdel, Leila; Pirdel, Manijeh

2014-06-01

This article presents an overview of the involvement of iron overload-induced nitric oxide (NO) overproduction in apoptosis of peritoneal macrophages of women with endometriosis. We have postulated that the peritoneal iron overload originated from retrograde menstruation or bleeding lesions in the ectopic endometrium, which may contribute to the development of endometriosis by a wide range of mechanisms, including oxidative damage and chronic inflammation. Excessive NO production may also be associated with impaired clearance of endometrial cells by macrophages, which promotes cell growth in the peritoneal cavity. Therefore, further research of the mechanisms and consequences of macrophage apoptosis in endometriosis helps discover novel therapeutic strategies that are designed to prevent progression of endometriosis. © 2014 Society for Reproduction and Fertility.

Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

PubMed

Gupta, Shishir Kumar; Yadav, Pavan Kumar; Gandham, Ravi Kumar; Sahoo, A P; Harish, D R; Singh, Arvind Kumar; Tiwari, A K

2016-02-02

Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity. Copyright © 2015 Elsevier B.V. All rights reserved.

17β-Estradiol regulates cell proliferation, colony formation, migration, invasion and promotes apoptosis by upregulating miR-9 and thus degrades MALAT-1 in osteosarcoma cell MG-63 in an estrogen receptor-independent manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Dengfeng; Yang, Hui; Lin, Jing

2015-02-20

In bone, different concentration of estrogen leads to various of physiological processes in osteoblast, such as the proliferation, migration, and apoptosis in an estrogen receptor-dependent manner. But little was known about the estrogen effects on osteosarcoma (OS). In this study, OS cell MG-63 was treated with low (1 nM) or high (100 nM) dose of 17β-Estradiol (E2) with the presence or absence of estrogen receptor α (ERα), for evaluating the E2 effects on proliferation, migration, invasion, colony formation and apoptosis. Consistent with a previous study, high dose of E2 treatment dramatically downregulated expressing level of long non-coding RNA metastasis associated lung adenocarcinomamore » transcript 1 (MALAT-1). The observation of upregulation of miR-9 after a high dose of E2 treatment indicated the cause of MALAT-1 reduction. Downregulation of MALAT-1 promoted the combination of SFPQ/PTBP2 complex. It was also observed that the proliferation, migration, invasion, colony formation and apoptosis of OS cells were remarkably affected by high dose of E2 treatment, but not by low dose, in an ERα independent manner. Furthermore, the abolishment of the effects on these physiological processes caused by ectopic expression of miR-9 ASOs suggested the necessity of miR-9 in MALAT-1 regulation. Here we found that the high dose of E2 treatment upregulated miR-9 thus posttranscriptionally regulated MALAT-1 RNA level in OS cells, and then the downregulation of MALAT-1 inhibited cell proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) processes in the E2-dose dependent and ER-independent ways. - Highlights: • E2 affects osteosarcoma cell MG-63 in an Estrogen receptor-independent way. • High dose of E2 treatment upregulates miR-9 which target to MALAT-1 RNA. • Upregulated miR-9 degrades MALAT-1 and thus affects combination of SFPQ/PTBP2. • E2 treatment block cell proliferation, colony formation, mobility, and enhance apoptosis.« less

Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

2011-01-21

Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

Transport via SLC5A8 with Subsequent Inhibition of Histone Deacetylases HDAC1 and HDAC3 Underlies the Antitumor Activity of 3-Bromopyruvate

PubMed Central

Thangaraju, Muthusamy; Karunakaran, Senthil K.; Itagaki, Shiro; Gopal, Elangovan; Elangovan, Selvakumar; Prasad, Puttur D.; Ganapathy, Vadivel

2009-01-01

Background 3-Bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of ATP production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The present studies have uncovered a new and novel mechanism for the antitumor activity of 3-bromopyruvate. Methods Transport of 3-bromopyruvate via SLC5A8, a tumor suppressor and a Na+-coupled electrogenic transporter for short-chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3-bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by FACS analysis and colony formation assay. Acetylation status of histone H4 was evaluated by Western blot. Results 3-Bromopyruvate is a transportable substrate for SLC5A8, with the transport process being Na+-coupled and electrogenic. MCF7 cells do not express SLC5A8 and are not affected by 3-bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells undergo apoptosis in the presence of 3-bromopyruvate. This cell death is associated with inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identify HDAC1 and HDAC3 as the targets for 3-bromopyruvate. Conclusions 3-Bromopyruvate is transported into cells actively via the tumor suppressor SLC5A8 and the process is energized by an electrochemical Na+ gradient. Ectopic expression of the transporter in MCF7 cells leads to apoptosis, and the mechanism involves inhibition of HDAC1/HDAC3. PMID:19637353

TP53INP1 is a novel p73 target gene that induces cell cycle arrest and cell death by modulating p73 transcriptional activity.

PubMed

Tomasini, Richard; Seux, Mylène; Nowak, Jonathan; Bontemps, Caroline; Carrier, Alice; Dagorn, Jean-Charles; Pébusque, Marie-Josèphe; Iovanna, Juan L; Dusetti, Nelson J

2005-12-08

TP53INP1 is an alternatively spliced gene encoding two nuclear protein isoforms (TP53INP1alpha and TP53INP1beta), whose transcription is activated by p53. When overexpressed, both isoforms induce cell cycle arrest in G1 and enhance p53-mediated apoptosis. TP53INP1s also interact with the p53 gene and regulate p53 transcriptional activity. We report here that TP53INP1 expression is induced during experimental acute pancreatitis in p53-/- mice and in cisplatin-treated p53-/- mouse embryo fibroblasts (MEFs). We demonstrate that ectopic expression of p73, a p53 homologue, leads to TP53INP1 induction in p53-deficient cells. In turn, TP53INP1s alters the transactivation capacity of p73 on several p53-target genes, including TP53INP1 itself, demonstrating a functional association between p73 and TP53INP1s. Also, when overexpressed in p53-deficient cells, TP53INP1s inhibit cell growth and promote cell death as assessed by cell cycle analysis and colony formation assays. Finally, we show that TP53INP1s potentiate the capacity of p73 to inhibit cell growth, that effect being prevented when the p53 mutant R175H is expressed or when p73 expression is blocked by a siRNA. These results suggest that TP53INP1s are functionally associated with p73 to regulate cell cycle progression and apoptosis, independently from p53.

Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

PubMed

Mozer, B A

2001-05-15

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. Copyright 2001 Academic Press.

ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

PubMed Central

Hida, Shigeaki; Fujii, Chifumi; Taniguchi, Shun’ichiro; Ito, Kensuke; Matsumura, Tomio; Okada, Nagisa; Sakaizawa, Takashi; Kobayashi, Akira; Takeoka, Michiko; Miyagawa, Shin-ichi

2017-01-01

ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy. PMID:28056049

N,N-dimethyl phytosphingosine induces caspase-8-dependent cytochrome c release and apoptosis through ROS generation in human leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Byeong Mo; Choi, Yun Jung; Han, Youngsoo

2009-08-15

N,N-dimethyl phytosphingosine (DMPS) blocks the conversion of sphingosine to sphingosine-1-phosphate (S1P) by the enzyme sphingosine kinase (SK). In this study, we elucidated the apoptotic mechanisms of DMPS action on a human leukemia cell line using functional pharmacologic and genetic approaches. First, we demonstrated that DMPS-induced apoptosis is evidenced by nuclear morphological change, distinct internucleosomal DNA fragmentation, and an increased sub-G1 cell population. DMPS treatment led to the activation of caspase-9 and caspase-3, accompanied by the cleavage of poly(ADP-ribose) polymerase (PARP) and led to cytochrome c release, depolarization of the mitochondrial membrane potential, and downregulation of the anti-apoptotic members of themore » bcl-2 family. Ectopic expression of bcl-2 and bcl-xL conferred resistance of HL-60 cells to DMPS-induced cell death, suggesting that DMPS-induced apoptosis occurs predominantly through the activation of the intrinsic mitochondrial pathway. We also observed that DMPS activated the caspase-8-Bid-Bax pathway and that the inhibition of caspase-8 by z-IETD-fmk or small interfering RNA suppressed the cleavage of Bid, cytochrome c release, caspase-3 activation, and apoptotic cell death. In addition, cells subjected to DMPS exhibited significantly increased reactive oxygen species (ROS) generation, and ROS scavengers, such as quercetin and Tiron, but not N-acetylcysteine (NAC), inhibited DMPS-induced activations of caspase-8, -3 and subsequent apoptotic cell death, indicating the role of ROS in caspase-8-mediated apoptosis. Taken together, these results indicate that caspase-8 acts upstream of caspase-3, and that the caspase-8-mediated mitochondrial pathway is important in DMPS-induced apoptosis. Our results also suggest that ROS are critical regulators of caspase-8-mediated apoptosis in DMPS-treated leukemia cells.« less

MicroRNA-520g Confers Drug Resistance by Regulating p21 Expression in Colorectal Cancer*

PubMed Central

Zhang, Yang; Geng, Liying; Talmon, Geoffrey; Wang, Jing

2015-01-01

Development of drug resistance is one of the major causes of colorectal cancer recurrence, yet mechanistic understanding and therapeutic options remain limited. Here, we show that expression of microRNA (miR)-520g is correlated with drug resistance of colon cancer cells. Ectopic expression of miR-520g conferred resistance to 5-fluorouracil (5-FU)- or oxaliplatin-induced apoptosis in vitro and reduced the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicated that miR-520g mediated drug resistance through down-regulation of p21 expression. Moreover, p53 suppressed miR-520g expression, and deletion of p53 up-regulated miR-520g expression. Inhibition of miR-520g in p53−/− cells increased their sensitivity to 5-FU treatment. Importantly, studies of patient samples indicated that expression of miR-520g correlated with chemoresistance in colorectal cancer. These findings indicate that the p53/miR-520g/p21 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of miR-520g or restoration of p21 expression may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, especially in those with mutant p53. PMID:25616665

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

PubMed Central

Chiappetta, A.; Fambrini, M.; Petrarulo, M.; Rapparini, F.; Michelotti, V.; Bruno, L.; Greco, M.; Baraldi, R.; Salvini, M.; Pugliesi, C.; Bitonti, M. B.

2009-01-01

Background and Aims The clone EMB-2 of the interspecific hybrid Helianthus annuus × H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. Methods Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography–mass spectrometry–selected ion monitoring method and an immuno-cytochemical approach. Key Results Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. Conclusions In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone. PMID:19151043

BIM is the primary mediator of MYC-induced apoptosis in multiple solid tissues.

PubMed

Muthalagu, Nathiya; Junttila, Melissa R; Wiese, Katrin E; Wolf, Elmar; Morton, Jennifer; Bauer, Barbara; Evan, Gerard I; Eilers, Martin; Murphy, Daniel J

2014-09-11

MYC is one of the most frequently overexpressed oncogenes in human cancer, and even modestly deregulated MYC can initiate ectopic proliferation in many postmitotic cell types in vivo. Sensitization of cells to apoptosis limits MYC's oncogenic potential. However, the mechanism through which MYC induces apoptosis is controversial. Some studies implicate p19ARF-mediated stabilization of p53, followed by induction of proapoptotic BH3 proteins NOXA and PUMA, whereas others argue for direct regulation of BH3 proteins, especially BIM. Here, we use a single experimental system to systematically evaluate the roles of p19ARF and BIM during MYC-induced apoptosis, in vitro, in vivo, and in combination with a widely used chemotherapeutic, doxorubicin. We find a common specific requirement for BIM during MYC-induced apoptosis in multiple settings, which does not extend to the p53-responsive BH3 family member PUMA, and find no evidence of a role for p19ARF during MYC-induced apoptosis in the tissues examined. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Adalimumab Treatment in Biologically Naïve Crohn's Disease: Relationship with Ectopic MUC5AC Expression and Endoscopic Improvement

PubMed Central

Mizoshita, Tsutomu; Tanida, Satoshi; Tsukamoto, Hironobu; Ozeki, Keiji; Katano, Takahito; Nishiwaki, Hirotaka; Ebi, Masahide; Mori, Yoshinori; Kubota, Eiji; Kataoka, Hiromi; Kamiya, Takeshi; Joh, Takashi

2014-01-01

Background. Adalimumab (ADA) is effective for patients with Crohn's disease (CD). However, there have been few reports on ADA therapy with respect to its relationship with pathologic findings and drug efficacy in biologically naïve CD cases. Methods. Fifteen patients with active biologically naïve CD were treated with ADA. We examined them clinically and pathologically with ectopic MUC5AC expression in the lesions before and after 12 and 52 weeks of ADA therapy, retrospectively. Results. Both mean CD activity index scores and serum C-reactive protein values were significantly lower after ADA therapy (P < 0.001). In the MUC5AC negative group, all cases exhibited clinical remission (CR) and endoscopic improvement at 52 weeks. In MUC5AC positive groups, loss of MUC5AC expression was detected in cases having CR and endoscopic improvement at 52 weeks, while remnant ectopic MUC5AC expression was observed in those exhibiting no endoscopic improvement and flare up after 52 weeks. Conclusions. ADA leads to CR and endoscopic improvement in biologically naïve CD cases. In addition, ectopic MUC5AC expression may be a predictive marker of flare up and endoscopic improvement in the intestines of CD patients. PMID:24829572

The serum miR-21 level serves as a predictor for the chemosensitivity of advanced pancreatic cancer, and miR-21 expression confers chemoresistance by targeting FasL.

PubMed

Wang, Peng; Zhuang, Liping; Zhang, Juan; Fan, Jie; Luo, Jianmin; Chen, Hao; Wang, Kun; Liu, Luming; Chen, Zhen; Meng, Zhiqiang

2013-06-01

miR-21 expression in cancer tissue has been reported to be associated with the clinical outcome and activity of gemcitabine in pancreatic cancer. However, resection is possible in only a minority of patients due to the advanced stages often present at the time of diagnosis, and safely obtaining sufficient quantities of pancreatic tumor tissue for molecular analysis is difficult at the unresectable stages. In this study, we investigated whether the serum level of miR-21 could be used as a predictor of chemosensitivity. We tested the levels of serum miR-21 in a cohort of 177 cases of advanced pancreatic cancer who received gemcitabine-based palliative chemotherapy. We found that a high level of miR-21 in the serum was significantly correlated with a shortened time-to-progression (TTP) and a lower overall survival (OS). The serum miR-21 level was an independent prognostic factor for both the TTP and the OS (HR 1.920; 95% CI, 1.274-2.903, p = 0.002 for TTP and HR 1.705; 95% CI, 1.147-2.535, p = 0.008 for OS). The results from a functional study showed that gemcitabine exposure down-regulated miR-21 expression and up-regulated FasL expression. The increased FasL expression following gemcitabine treatment induced cancer cell apoptosis, whereas the ectopic expression of miR-21 partially protected the cancer cells from gemcitabine-induced apoptosis. Additionally, we confirmed that FasL was a direct target of miR-21. Therefore, the serum level of miR-21 may serve as a predictor of chemosensitivity in advanced pancreatic cancer. Additionally, we identified a new mechanism of chemoresistance mediated by the effects of miR-21 on the FasL/Fas pathway. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Spermatogenesis arrest caused by conditional deletion of Hsp90α in adult mice

PubMed Central

Kajiwara, Chiaki; Kondo, Shiho; Uda, Shizuha; Dai, Lei; Ichiyanagi, Tomoko; Chiba, Tomoki; Ishido, Satoshi; Koji, Takehiko; Udono, Heiichiro

2012-01-01

Summary It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90α is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90α and found that conditional deletion of Hsp90α in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90α KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90α KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90α in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty. PMID:23213375

Ectopic expression of Msx2 in mammalian myotubes recapitulates aspects of amphibian muscle dedifferentiation.

PubMed

Yilmaz, Atilgan; Engeler, Rachel; Constantinescu, Simona; Kokkaliaris, Konstantinos D; Dimitrakopoulos, Christos; Schroeder, Timm; Beerenwinkel, Niko; Paro, Renato

2015-11-01

In contrast to urodele amphibians and teleost fish, mammals lack the regenerative responses to replace large body parts. Amphibian and fish regeneration uses dedifferentiation, i.e., reversal of differentiated state, as a means to produce progenitor cells to eventually replace damaged tissues. Therefore, induced activation of dedifferentiation responses in mammalian tissues holds an immense promise for regenerative medicine. Here we demonstrate that ectopic expression of Msx2 in cultured mouse myotubes recapitulates several aspects of amphibian muscle dedifferentiation. We found that MSX2, but not MSX1, leads to cellularization of myotubes and downregulates the expression of myotube markers, such as MHC, MRF4 and myogenin. RNA sequencing of myotubes ectopically expressing Msx2 showed downregulation of over 500 myotube-enriched transcripts and upregulation of over 300 myoblast-enriched transcripts. MSX2 selectively downregulated expression of Ptgs2 and Ptger4, two members of the prostaglandin pathway with important roles in myoblast fusion during muscle differentiation. Ectopic expression of Msx2, as well as Msx1, induced partial cell cycle re-entry of myotubes by upregulating CyclinD1 expression but failed to initiate S-phase. Finally, MSX2-induced dedifferentiation in mouse myotubes could be recapitulated by a pharmacological treatment with trichostatin A (TSA), bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 1 (FGF1). Together, these observations indicate that MSX2 is a major driver of dedifferentiation in mammalian muscle cells. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Hepatic Leukemia Factor Promotes Resistance To Cell Death: Implications For Therapeutics and Chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J.

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation.« less

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway

PubMed Central

Yanai, H; Yoshioka, Y; Yoshida, H; Nakao, Y; Plessis, A; Yamaguchi, M

2014-01-01

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis. PMID:24752236

Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway.

PubMed

Yanai, H; Yoshioka, Y; Yoshida, H; Nakao, Y; Plessis, A; Yamaguchi, M

2014-04-21

Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.

A nontranscriptional role for Oct4 in the regulation of mitotic entry

PubMed Central

Zhao, Rui; Deibler, Richard W.; Lerou, Paul H.; Ballabeni, Andrea; Heffner, Garrett C.; Cahan, Patrick; Unternaehrer, Juli J.; Kirschner, Marc W.; Daley, George Q.

2014-01-01

Rapid progression through the cell cycle and a very short G1 phase are defining characteristics of embryonic stem cells. This distinct cell cycle is driven by a positive feedback loop involving Rb inactivation and reduced oscillations of cyclins and cyclin-dependent kinase (Cdk) activity. In this setting, we inquired how ES cells avoid the potentially deleterious consequences of premature mitotic entry. We found that the pluripotency transcription factor Oct4 (octamer-binding transcription factor 4) plays an unappreciated role in the ES cell cycle by forming a complex with cyclin–Cdk1 and inhibiting Cdk1 activation. Ectopic expression of Oct4 or a mutant lacking transcriptional activity recapitulated delayed mitotic entry in HeLa cells. Reduction of Oct4 levels in ES cells accelerated G2 progression, which led to increased chromosomal missegregation and apoptosis. Our data demonstrate an unexpected nontranscriptional function of Oct4 in the regulation of mitotic entry. PMID:25324523

Alteration of Nrf2 and Glutamate Cysteine Ligase expression contribute to lesions growth and fibrogenesis in ectopic endometriosis.

PubMed

Marcellin, L; Santulli, P; Chouzenoux, S; Cerles, O; Nicco, C; Dousset, B; Pallardy, M; Kerdine-Römer, S; Just, P A; Chapron, C; Batteux, F

2017-09-01

The redox-sensitive nuclear factor erythroid-derived 2-like 2 (NRF2) controls endogenous antioxidant enzymes' transcription and protects against oxidative damage which is triggered by inflammation and known to favor progression of endometriosis. Glutamate Cysteine Ligase (GCL), a target gene of NRF2, is the first enzyme in the synthesis cascade of glutathione, an important endogenous antioxidant. Sixty-one patients, with thorough surgical examination of the abdominopelvic cavity, were recruited for the study: 31 with histologically-proven endometriosis and 30 disease-free women taken as controls. Expressions of NRF2 and GCL were investigated by quantitative RT-PCR and immunohistochemistry in eutopic and ectopic endometria from endometriosis-affected women and in endometrium of disease-free women. Ex vivo stromal and epithelial cells were extracted and purified from endometrial and endometriotic biopsies to explore expression of NRF2 and GCL in both stromal and epithelial compartments by western blot. Finally, in order to strengthen the role of NRF2 in endometriosis pathogenesis, we evaluated the drop of NRF2 expression in a mouse model of endometriosis using NRF2 knockout (NRF2 -/- ) mice. The mRNA levels of NRF2 and GCL were significantly lower in ectopic endometria of endometriosis-affected women compared to eutopic endometria of disease-free women. The immunohistochemical analysis confirmed the decreased expression of both NRF2 and GCL in ectopic endometriotic tissues compared to eutopic endometria of endometriosis-affected and disease-free women. Immunoblotting revealed a significant decreased of NRF2 and GCL expression in epithelial and stroma cells from ectopic lesions of endometriosis-affected women compared to eutopic endometria from controls. Using a murine model of endometriosis, NRF2 -/- implants were more fibrotic compared to wild-type with an increased weight and volume. These findings indicate that expression of the transcription factor NRF2 and its effector GCL are both profoundly deregulated in endometriotic lesions towards increased growth and fibrogenetic processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Compartmentalization of the somite and myogenesis in chick embryos are influenced by wnt expression.

PubMed

Wagner, J; Schmidt, C; Nikowits, W; Christ, B

2000-12-01

Muscles of the body and bones of the axial skeleton derive from specialized regions of somites. Somite development is influenced by adjacent structures. In particular, the dorsal neural tube and the overlying ectoderm have been shown to be necessary for the induction of myogenic precursor cells in the dermomyotome. Members of the Wnt family of signaling molecules, which are expressed in the dorsal neural tube and the ectoderm, are postulated to be responsible for this process. It is shown here that ectopically implanted Wnt-1-, -3a-, and -4-expressing cells alter the process of somite compartmentalization in vivo. An enlarged dorsal compartment results from the implantation of Wnt-expressing cells ventrally between the neural tube/notochord and epithelial somites, at the expense of the ventral compartment, the sclerotome. Thus, ectopic Wnt expression is able to override the influence of ventralizing signals arising from notochord and floor plate. This shift of the border between the two compartments was identified by an increase in the domain of Pax-3 expression and a complete loss of Pax-1 expression in somites close to the ectopic Wnt signal. The expanded expression of MyoD and desmin provides evidence that it is the myotome which increases as a result of Wnt signaling. Paraxis expression is also drastically amplified after implantation of Wnt-expressing cells indicating that Wnts are involved in the formation and maintenance of somite epithelium and suggesting that Paraxis is activated through Wnt signaling pathways. Taken together these results suggest that ectopic Wnts disturb the normal balance of signaling molecules within the somite, resulting in an enhanced recruitment of somitic cells into the myogenic lineage. Copyright 2000 Academic Press.

Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin

PubMed Central

Kortmann, Jens; Brubaker, Sky W.

2015-01-01

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6. PMID:26109648

DEAD-box helicase 27 promotes colorectal cancer growth and metastasis and predicts poor survival in CRC patients.

PubMed

Tang, Jieting; Chen, Huarong; Wong, Chi-Chun; Liu, Dabin; Li, Tong; Wang, Xiaohong; Ji, Jiafu; Sung, Joseph Jy; Fang, Jing-Yuan; Yu, Jun

2018-03-14

Copy number alterations (CNAs) are crucial for colorectal cancer (CRC) development. In this study, DEAD box polypeptide 27 (DDX27) was identified to be highly amplified in both TCGA CRC (474/615) and primary CRC (47/103), which was positively correlated with its mRNA overexpression. High DDX27 mRNA (N = 199) and protein expression (N = 260) predicted poor survival in CRC patients. Ectopic expression of DDX27 increased CRC cells proliferation, migration and invasion, but suppressed apoptosis. Conversely, silencing of DDX27 exerted opposite effects in vitro and significantly inhibited murine xenograft tumor growth and lung metastasis in vivo. Up-regulation of DDX27 enhanced and prolonged TNF-α-mediated NF-κB signaling. Nucleophosmin (NPM1) was identified as a binding partner of DDX27. DDX27 increased nuclear NPM1 and NF-κB-p65 interaction to enhance DNA binding activity of NF-κB. Silencing NPM1 abrogated DDX27-activating NF-κB signaling and its tumor-promoting function. Together, DDX27 is overexpressed and plays a pivotal oncogenic role in CRC.

Enhanced Development of Skeletal Myotubes from Porcine Induced Pluripotent Stem Cells

PubMed Central

Genovese, Nicholas J.; Domeier, Timothy L.; Telugu, Bhanu Prakash V. L.; Roberts, R. Michael

2017-01-01

The pig is recognized as a valuable model in biomedical research in addition to its agricultural importance. Here we describe a means for generating skeletal muscle efficiently from porcine induced pluripotent stem cells (piPSC) in vitro thereby providing a versatile platform for applications ranging from regenerative biology to the ex vivo cultivation of meat. The GSK3B inhibitor, CHIR99021 was employed to suppress apoptosis, elicit WNT signaling events and drive naïve-type piPSC along the mesoderm lineage, and, in combination with the DNA methylation inhibitor 5-aza-cytidine, to activate an early skeletal muscle transcription program. Terminal differentiation was then induced by activation of an ectopically expressed MYOD1. Myotubes, characterized by myofibril development and both spontaneous and stimuli-elicited excitation-contraction coupling cycles appeared within 11 days. Efficient lineage-specific differentiation was confirmed by uniform NCAM1 and myosin heavy chain expression. These results provide an approach for generating skeletal muscle that is potentially applicable to other pluripotent cell lines and to generating other forms of muscle. PMID:28165492

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latonen, Leena; Jaervinen, Paeivi M.; Haartman Institute, University of Helsinki, FIN-00014 Helsinki

2008-02-15

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1more » correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions.« less

Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis.

PubMed

Ko, Hyun-Ja; Kinkel, Sarah A; Hubert, François-Xavier; Nasa, Zeyad; Chan, James; Siatskas, Christopher; Hirubalan, Premila; Toh, Ban-Hock; Scott, Hamish S; Alderuccio, Frank

2010-12-01

The autoimmune regulator (AIRE) promotes "promiscuous" expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG(35-55), transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Sujun; Southern Medical University, Guangzhou, Guangdong 510515; Wu, Binwen, E-mail: wubinwengd@aliyun.com

2014-02-14

Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 andmore » the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.« less

miR-338-3p confers 5-fluorouracil resistance in p53 mutant colon cancer cells by targeting the mammalian target of rapamycin.

PubMed

Han, Jia; Li, Jie; Tang, Kaijie; Zhang, Huahua; Guo, Bo; Hou, Ni; Huang, Chen

2017-11-15

Evidence demonstrate that p53 mutations and microRNAs (miRs) are important components of 5-FU resistance in colorectal cancer (CRC). miR-338-3p has been reported associated with cancer prognosis. However whether or not it influences chemotherapy sensitivity and the underlying mechanisms have not been elucidated. Here, three types of human colon cancer cell lines, HT29 (mutant p53), HCT116 (wild-type p53), and HCT116 p53 -/- (deficient p53), were treated with 5-FU. We showed that expression of miR-338-3p was correlated with apoptosis and 5-FU resistance in colon cancer cells. Ectopic expression of miR-338-3p conferred resistance to 5-FU in HCT116 cells. Further experiments indicated that miR-338-3p mediated 5-FU resistance through down-regulation of mTOR expression. Moreover, inhibition of miR-338-3p in HT29 and HCT116 p53 -/- cells increased their sensitivity to 5-FU treatment. Furthermore, we detected autophagy changes in our experiment because mTOR was known prominently regulating autophagy and the competition between autophagy and apoptosis in response to 5-FU was a mechanism influencing 5-FU sensitivity. Our results reveal a critical and novel role of miR-338-3p in the correlation of 5-FU resistance with p53 status. Moreover, the miR-338-3p inhibitor has the potential to overcome 5-FU resistance in p53 mutant colon cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Hormonal therapy deregulates prostaglandin-endoperoxidase synthase 2 (PTGS2) expression in endometriotic tissues.

PubMed

Santulli, Pietro; Borghese, Bruno; Noël, Jean-Christophe; Fayt, Isabelle; Anaf, Vincent; de Ziegler, Dominique; Batteux, Frederic; Vaiman, Daniel; Chapron, Charles

2014-03-01

Endometriosis is a common gynecologic condition characterized by an important inflammatory process mediated by the prostaglandin pathway. Oral contraceptives are the treatment of choice for symptomatic endometriotic women. However the effects of oral contraceptives use and prostaglandin pathway in endometriotic women are actually still unknown. To investigate the expression of prostaglandin pathway key genes in endometriotic tissue, affected or not by hormonal therapy, as compared with healthy endometrial tissue. This was a comparative laboratory study. This study was conducted in a tertiary-care university hospital. Seventy-six women, with (n = 46) and without (n = 30) histologically proven endometriosis. Prostaglandin-endoperoxidase synthase (PTGS)1, PTGS2, prostaglandin E receptor (PTGER)1, PTGER2, PTGER3, and PTGER4 mRNA levels in endometrium of disease-free women and in eutopic and ectopic endometrium of endometriosis-affected women. PTGS2 expression was further investigated by immunohistochemistry, using specific monoclonal antibodies. PTGS2 expression was analyzed at mRNA and protein levels and correlated with taking hormonal treatment. PTGS2 expression was significantly increased in eutopic and ectopic endometrium as compared with healthy tissue (induction of 9.6- and 6.3-fold, respectively; P = .001). PTGS2 immunoreactivity increased gradually from normal endometrium to eutopic and ectopic endometrium (h-score of 96.7 ± 55.0, 128.3 ± 66.1, and 226.7 ± 62.6, respectively, P < .001). PTGER2, PTGER3, and PTGER4 expression increased significantly and gradually from normal to eutopic and ectopic endometrium, whereas PTGER1 remained unchanged. Patients under hormonal treatment had a higher PTGS2 expression at transcriptional and protein levels as compared with those without treatment (P = .002 and P = .025, respectively). Prostaglandin pathway is strongly deregulated in eutopic and ectopic endometrium of women suffering from endometriosis for the benefit of an increased PTGS2 expression. We show for the first time that hormonal treatment appears to enhance even more PTGS2 expression. These results contribute to explain why medical treatment could fail to control endometriosis progression.

Cloning and functional characterization of the guinea pig apoptosis inhibitor protein Survivin.

PubMed

Habtemichael, Negusse; Wünsch, Desiree; Bier, Carolin; Tillmann, Sarah; Unruhe, Britta; Frauenknecht, Katrin; Heinrich, Ulf-Rüdiger; Mann, Wolf J; Stauber, Roland H; Knauer, Shirley K

2010-12-01

The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (Survivin(Gp)) are currently lacking. Here, we here report the cloning and functional characterization of Survivin(Gp). The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. Survivin(Gp) displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that Survivin(Gp) is more related to humans than to rodents. Ectopic expression studies of a Survivin(Gp)-GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, Survivin(Gp)-GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for Survivin(Gp)-GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of Survivin(Gp) during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that Survivin(Gp) can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as spleen and liver, we also found Survivin in terminally differentiated cell types. Importantly, Survivin was detectable also in the cochlea, suggesting a potential role for Survivin in the auditory system. We provide the first experimental evidence for the expression of Survivin in the guinea pig. As Survivin(Gp) can substitute for known functions of human Survivin, the guinea pig model will now also allow investigating Survivin's (patho)physiological role and to test Survivin-directed potential therapeutic strategies. Copyright © 2010 Elsevier B.V. All rights reserved.

Ectopic expression of hoxb2 after retinoic acid treatment or mRNA injection: disruption of hindbrain and craniofacial morphogenesis in zebrafish embryos.

PubMed

Yan, Y L; Jowett, T; Postlethwait, J H

1998-12-01

To investigate pattern formation in the vertebrate hindbrain, we isolated a full length hoxb2 cDNA clone from zebrafish. In a gene phylogeny, zebrafish hoxb2 clusters with human HOXB2, and it maps on linkage group 3 along with several other loci whose orthologues are syntenic with human HOXB2. In the hindbrain, hoxb2 is expressed at high levels in rhombomere 3 (r3), lower levels in r4, still lower in r5, and at undetectable levels in r6. In r7, r8, and the rostral spinal cord, hoxb2 is expressed at a lower level than in r5. Lateral cells appearing to emanate from r4 express both hoxb2 and dlx2, suggesting that they are neural crest. Overexpression of hoxb2 by mRNA injections into early cleavage stage embryos resulted in abnormal morphogenesis of the midbrain and rostral hindbrain, abnormal patterning in r4, fusion of cartilage elements arising from pharyngeal arches 1 and 2, and ectopic expression of krx20 and valentino (but not pax2, rtk1, or hoxb1) in the rostral hindbrain, midbrain, and, surprisingly, the eye. Treatments with retinoic acid produced a phenotype similar to that of ectopic hoxb2 expression, including ectopic krx20 (but not valentino) expression in the eye, and fusion of cartilages from pharyngeal arches 1 and 2. The results suggest that hoxb2 plays an important role in the patterning of hindbrain and pharyngeal arches in the zebrafish.

The timecourse of apoptotic cell death during postnatal remodeling of the mouse cochlea and its premature onset by triiodothyronine (T3).

PubMed

Peeters, R P; Ng, L; Ma, M; Forrest, D

2015-05-15

Apoptosis underlies various forms of tissue remodeling during development. Prior to the onset of hearing, thyroid hormone (T3) promotes cochlear remodeling, which involves regression of the greater epithelial ridge (GER), a transient structure of columnar cells adjacent to the mechanosensory hair cells. We investigated the timecourse of apoptosis in the GER and the influence of ectopic T3 on apoptosis. In saline-treated mice, activated caspase 3-positive cells were detected in the GER between postnatal days 7 and 13 and appeared progressively along the cochlear duct from base to apex over developmental time. T3 given on P0 and P1 advanced the overall program of apoptosis and remodeling by ~4 days. Thyroid hormone receptor β was required for these actions, suggesting a receptor-mediated process of initiation of apoptosis. Finally, T3 given only at P0 or P1 resulted in deafness in adult mice, thus revealing a transient period of susceptibility to long-term damage in the neonatal auditory system. Published by Elsevier Ireland Ltd.

YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

PubMed Central

Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B.

2012-01-01

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity. PMID:22563435

YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense.

PubMed

Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B

2012-01-01

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.

Downregulation of miRNA-30c and miR-203a is associated with hepatitis C virus core protein-induced epithelial–mesenchymal transition in normal hepatocytes and hepatocellular carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Dongjing; Wu, Jilin, E-mail: 6296082@qq.com; Liu, Meizhou

Hepatitis C virus (HCV) Core protein has been demonstrated to induce epithelial–mesenchymal transition (EMT) and is associated with cancer progression of hepatocellular carcinoma (HCC). However, how the Core protein regulates EMT is still unclear. In this study, HCV Core protein was overexpressed by an adenovirus. The protein levels of EMT markers were measured by Western blot. The xenograft animal model was established by inoculation of HepG2 cells. Results showed that ectopic expression of HCV core protein induced EMT in L02 hepatocytes and HepG2 tumor cells by upregulating vimentin, Sanl1, and Snal2 expression and downregulating E-cadherin expression. Moreover, Core protein downregulatedmore » miR-30c and miR-203a levels in L02 and HepG2 cells, but artificial expression of miR-30c and miR-203a reversed Core protein-induced EMT. Further analysis showed that ectopic expression of HCV core protein stimulated cell proliferation, inhibited apoptosis, and increased cell migration, whereas artificial expression of miR-30c and miR-203a significantly reversed the role of Core protein in these cell functions in L02 and HepG2 cells. In the HepG2 xenograft tumor models, artificial expression of miR-30c and miR-203a inhibited EMT and tumor growth. Moreover, L02 cells overexpressing Core protein can form tumors in nude mice. In HCC patients, HCV infection significantly shortened patients' survival time, and loss of miR-30c and miR-203 expression correlated with poor survival. In conclusion, HCV core protein downregulates miR-30c and miR-203a expression, which results in activation of EMT in normal hepatocytes and HCC tumor cells. The Core protein-activated-EMT is involved in the carcinogenesis and progression of HCC. Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC. - Highlights: • HCV core protein downregulates miR-30c and miR-203a expression. • Downregulation of miR-30c and miR-203a activates EMT. • Activated-EMT is involved in the carcinogenesis and progression of HCC. • Loss of miR-30c and miR-203a expression is a marker for the poor prognosis of HCC.« less

The role of Sox6 in zebrafish muscle fiber type specification.

PubMed

Jackson, Harriet E; Ono, Yosuke; Wang, Xingang; Elworthy, Stone; Cunliffe, Vincent T; Ingham, Philip W

2015-01-01

The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation in the zebrafish, a role similar to that ascribed to its murine ortholog.

Increased expression of resistin in ectopic endometrial tissue of women with endometriosis.

PubMed

Oh, Yoon Kyung; Ha, Young Ran; Yi, Kyong Wook; Park, Hyun Tae; Shin, Jung-Ho; Kim, Tak; Hur, Jun-Young

2017-11-01

Inflammation is a key process in the establishment and progression of endometriosis. Resistin, an adipocytokine, has biological properties linked to immunologic functions, but its role in endometriosis is unclear. Resistin gene expression was examined in eutopic and ectopic endometrial tissues from women with (n=25) or without (n=25) endometriosis. Resistin mRNA and protein levels were determined in endometrial tissue using quantitative real-time reverse transcription PCR and Western blotting, following adipokine profiling arrays. Resistin protein was detected in human endometrial tissues using an adipokine array test. Resistin mRNA and protein levels were significantly higher in ectopic endometrial tissue of patients with endometriosis than in normal eutopic endometrial tissue. Our results indicate that resistin is differentially expressed in endometrial tissues from women with endometriosis and imply a role for resistin in endometriosis-associated pelvic inflammation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ionizing Radiation–Inducible miR-27b Suppresses Leukemia Proliferation via Targeting Cyclin A2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Bo; Li, Dongping; Kovalchuk, Anna

2014-09-01

Purpose: Ionizing radiation is a common carcinogen that is important for the development of leukemia. However, the underlying epigenetic mechanisms remain largely unknown. The goal of the study was to explore microRNAome alterations induced by ionizing radiation (IR) in murine thymus, and to determine the role of IR-inducible microRNA (miRNA/miR) in the development of leukemia. Methods and Materials: We used the well-established C57BL/6 mouse model and miRNA microarray profiling to identify miRNAs that are differentially expressed in murine thymus in response to irradiation. TIB152 human leukemia cell line was used to determine the role of estrogen receptor–α (ERα) in miR-27bmore » transcription. The biological effects of ectopic miR-27b on leukemogenesis were measured by western immunoblotting, cell viability, apoptosis, and cell cycle analyses. Results: Here, we have shown that IR triggers the differential expression of miR-27b in murine thymus tissue in a dose-, time- and sex-dependent manner. miR-27b was significantly down-regulated in leukemia cell lines CCL119 and TIB152. Interestingly, ERα was overexpressed in those 2 cell lines, and it was inversely correlated with miR-27b expression. Therefore, we used TIB152 as a model system to determine the role of ERα in miR-27b expression and the contribution of miR-27b to leukemogenesis. β-Estradiol caused a rapid and transient reduction in miR-27b expression reversed by either ERα-neutralizing antibody or ERK1/2 inhibitor. Ectopic expression of miR-27b remarkably suppressed TIB152 cell proliferation, at least in part, by inducing S-phase arrest. In addition, it attenuated the expression of cyclin A2, although it had no effect on the levels of PCNA, PPARγ, CDK2, p21, p27, p-p53, and cleaved caspase-3. Conclusion: Our data reveal that β-estradiol/ERα signaling may contribute to the down-regulation of miR-27b in acute leukemia cell lines through the ERK1/2 pathway, and that miR-27b may function as a tumor suppressor that inhibits cell proliferation by targeting cyclin A2.« less

Expression and regulation of estrogen-converting enzymes in ectopic human endometrial tissue.

PubMed

Fechner, Sabine; Husen, Bettina; Thole, Hubert; Schmidt, Markus; Gashaw, Isabella; Kimmig, Rainer; Winterhager, Elke; Grümmer, Ruth

2007-10-01

To investigate the regulation of estrogen-converting enzymes in human ectopic endometrial tissue. Animal study. Academic medical center. Sixty female nude mice with implanted human endometrial tissue. Twenty-two premenopausal women undergoing endometrial biopsy or hysterectomy. Human endometrial tissue was implanted into the peritoneal cavity of nude mice, and the effect of therapeutic drugs on transcription of steroid receptors and estrogen-converting enzymes was analyzed. Transcript levels of steroid hormone receptors, 17beta-hydroxysteroid dehydrogenase type 1 and 2, aromatase, and steroid sulfatase as well as proliferation rate were analyzed in the human ectopic endometrial tissue. Steroid receptors and estrogen-converting enzymes were expressed in the ectopic human endometrial fragments. Application of medroxyprogesterone acetate, dydrogesterone, danazol, and the aromatase inhibitor finrozole significantly inhibited aromatase transcription. In addition, danazol caused a significant decrease in transcription of steroid sulfatase, and finrozole, of 17beta-hydroxysteroid dehydrogenase type 1 in parallel to a decrease in proliferation rate in the ectopic human endometrial tissue. Pharmacological regulation of transcription of estrogen-converting enzymes in human endometrium cultured in nude mice may help to develop new therapeutic concepts based on local regulation of estrogen metabolism in endometriosis.

High Expression of High-Mobility Group Box 1 in Menstrual Blood: Implications for Endometriosis.

PubMed

Shimizu, Keiko; Kamada, Yasuhiko; Sakamoto, Ai; Matsuda, Miwa; Nakatsuka, Mikiya; Hiramatsu, Yuji

2017-11-01

Endometriosis is a benign gynecologic disease characterized by the presence of ectopic endometrium and associated with inflammation and immune abnormalities. However, the molecular basis for endometriosis is not well understood. To address this issue, the present study examined the expression of high-mobility group box (HMGB) 1 in menstrual blood to investigate its role in the ectopic growth of human endometriotic stromal cells (ESCs). A total of 139 patients were enrolled in this study; 84 had endometriosis and 55 were nonendometriotic gynecological patients (control). The HMGB1 levels in various fluids were measured by enzyme-linked immunosorbent assay. Expression of receptor for advanced glycation end products (RAGE) in eutopic and ectopic endometrium was assessed by immunohistochemistry, and RAGE and vascular endothelial growth factor ( VEGF) messenger RNA expression in HMGB1- and lipopolysaccharide (LPS)-treated ESCs was evaluated by real-time polymerase chain reaction. The HMGB1 concentration was higher in menstrual blood than in serum or peritoneal fluid ( P < .001 for both). RAGE was expressed in both normal and ectopic endometrium. Administration of 1000 ng/mL HMGB1 or coadministration of 100 ng/mL HMGB1 and 100 ng/mL LPS induced VEGF production in ESCs relative to the control ( P < .05). These results suggest that menstrual fluid has naturally high levels of HMGB1 and may promote endometriosis following retrograde menstruation when complexed with other factors such as LPS by inducing inflammation and angiogenesis.

A combination of a ribonucleotide reductase inhibitor and histone deacetylase inhibitors downregulates EGFR and triggers BIM-dependent apoptosis in head and neck cancer

PubMed Central

Habtemichael, Negusse; Bier, Carolin; Unruhe, Britta; Weisheit, Simona; Spange, Stephanie; Nonnenmacher, Frank; Fetz, Verena; Ginter, Torsten; Reichardt, Sigrid; Liebmann, Claus; Schneider, Günter; Krämer, Oliver H.

2012-01-01

Head and neck squamous cell carcinomas (HNSCCs) are the sixth most common malignant neoplasm and more than 50% of patients succumb to this disease. HNSCCs are characterized by therapy resistance, which relies on the overexpression of anti-apoptotic proteins and on the aberrant regulation of the epidermal growth factor receptor (EGFR). As inherent and acquired resistance to therapy counteracts improvement of long-term survival, novel multi-targeting strategies triggering cancer cell death are urgently required. We investigated how induction of replicational stress by the ribonucleotide reductase inhibitor hydroxyurea (HU) combined with histone deacetylase inhibitors (HDACi) exerts anti-tumor activity. We treated HNSCC cell lines and freshly isolated tumor cells with HDACi, such as the clinically approved anti-epileptic drug valproic acid (VPA), in combination with HU. Our data demonstrate that at clinically achievable levels VPA/HU combinations efficiently block proliferation as well as clonogenic survival, and trigger apoptosis of HNSCC cells. In the presence of VPA/HU, such tumor cells increase expression of the pro-apoptotic BCL-2 family protein BIM, independent of wild-type p53 signaling and in the absence of increased expression of the p53 targets PUMA and BAX. The pro-apoptotic activity of BIM in HNSCCs was found critical for tumor cell death; ectopic overexpression of BIM induced HNSCC apoptosis and RNAi-mediated depletion of BIM protected HNSCC cells from VPA/HU. Also, significantly elevated BIM levels (p<0.01) were detectable in the apoptotic tumor centers versus proliferating tumor margins in HNSCC patients (n=31), underlining BIM's clinical relevance. Importantly, VPA/HU treatment additionally reduces expression and cell surface localization of EGFR. Accordingly, in a xenograft mouse model, VPA/HU efficiently blocked tumor growth (P<0.001) correlating with BIM induction and EGFR downregulation. We provide a molecular rationale for the potent anti-cancer activities of this drug combination. Our data suggest its exploitation as a potential strategy for the treatment of HNSCC and other tumor entities characterized by therapy resistance linked to dysregulated EGFR activation. PMID:22289787

Mutations in the murine homologue of TUBB5 cause microcephaly by perturbing cell cycle progression and inducing p53-associated apoptosis.

PubMed

Breuss, Martin; Fritz, Tanja; Gstrein, Thomas; Chan, Kelvin; Ushakova, Lyubov; Yu, Nuo; Vonberg, Frederick W; Werner, Barbara; Elling, Ulrich; Keays, David A

2016-04-01

Microtubules play a crucial role in the generation, migration and differentiation of nascent neurons in the developing vertebrate brain. Mutations in the constituents of microtubules, the tubulins, are known to cause an array of neurological disorders, including lissencephaly, polymicrogyria and microcephaly. In this study we explore the genetic and cellular mechanisms that cause TUBB5-associated microcephaly by exploiting two new mouse models: a conditional E401K knock-in, and a conditional knockout animal. These mice present with profound microcephaly due to a loss of upper-layer neurons that correlates with massive apoptosis and upregulation of p53. This phenotype is associated with a delay in cell cycle progression and ectopic DNA elements in progenitors, which is dependent on the dosage of functional Tubb5. Strikingly, we report ectopic Sox2-positive progenitors and defects in spindle orientation in our knock-in mouse line, which are absent in knockout animals. This work sheds light on the functional repertoire of Tubb5, reveals that the E401K mutation acts by a complex mechanism, and demonstrates that the cellular pathology driving TUBB5-associated microcephaly is cell death. © 2016. Published by The Company of Biologists Ltd.

Post-Traumatic Osteoarthritis in Mice Following Mechanical Injury to the Synovial Joint

PubMed Central

Rai, Muhammad Farooq; Duan, Xin; Quirk, James D.; Holguin, Nilsson; Schmidt, Eric J.; Chinzei, Nobuaki; Silva, Matthew J.; Sandell, Linda J.

2017-01-01

We investigated the spectrum of lesions characteristic of post-traumatic osteoarthritis (PTOA) across the knee joint in response to mechanical injury. We hypothesized that alteration in knee joint stability in mice reproduces molecular and structural features of PTOA that would suggest potential therapeutic targets in humans. The right knees of eight-week old male mice from two recombinant inbred lines (LGXSM-6 and LGXSM-33) were subjected to axial tibial compression. Three separate loading magnitudes were applied: 6N, 9N, and 12N. Left knees served as non-loaded controls. Mice were sacrificed at 5, 9, 14, 28, and 56 days post-loading and whole knee joint changes were assessed by histology, immunostaining, micro-CT, and magnetic resonance imaging. We observed that tibial compression disrupted joint stability by rupturing the anterior cruciate ligament (except for 6N) and instigated a cascade of temporal and topographical features of PTOA. These features included cartilage extracellular matrix loss without proteoglycan replacement, chondrocyte apoptosis at day 5, synovitis present at day 14, osteophytes, ectopic calcification, and meniscus pathology. These findings provide a plausible model and a whole-joint approach for how joint injury in humans leads to PTOA. Chondrocyte apoptosis, synovitis, and ectopic calcification appear to be targets for potential therapeutic intervention. PMID:28345597

Phenotype anchoring in zebrafish reveals a potential role for matrix metalloproteinases (MMPs) in tamoxifen's effects on skin epithelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bugel, Sean M., E-mail: Sean.Bugel@oregonstate.edu; Wehmas, Leah C., E-mail: wehmasl@onid.oregonstate.edu; La Du, Jane K., E-mail: Jane.LaDu@oregonstate.edu

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen — a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥ 10 μM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustlymore » misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3–7 fold), consistent with AP-1 activity — a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin. - Highlights: • Tamoxifen rapidly induced a unique necrotic caudal fin phenotype in zebrafish. • Apoptosis co-localized temporally and spatially in the necrotic tail. • The necrotic fin phenotype was p53, GPER and ER independent. • The necrotic fin phenotype was dependent on ectopic MMP induction and activity in the skin. • The necrotic fin phenotype occurred at concentrations exceeding anti-estrogenic effects.« less

Transcriptional upregulation of p19INK4d upon diverse genotoxic stress is critical for optimal DNA damage response.

PubMed

Ceruti, Julieta M; Scassa, María E; Marazita, Mariela C; Carcagno, Abel C; Sirkin, Pablo F; Cánepa, Eduardo T

2009-06-01

p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.

Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3-bromopyruvate.

PubMed

Thangaraju, Muthusamy; Karunakaran, Senthil K; Itagaki, Shiro; Gopal, Elangovan; Elangovan, Selvakumar; Prasad, Puttur D; Ganapathy, Vadivel

2009-10-15

3-bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3-bromopyruvate. The transport of 3-bromopyruvate by sodium-coupled monocarboxylate transporter SMCT1 (SLC5A8), a tumor suppressor and a sodium (Na+)-coupled, electrogenic transporter for short-chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3-bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by fluorescence-activated cell-sorting analysis and colony-formation assay. The acetylation status of histone H4 was evaluated by Western blot analysis. 3-Bromopyruvate is a transportable substrate for SLC5A8, and that transport process is Na+-coupled and electrogenic. MCF7 cells did not express SLC5A8 and were not affected by 3-bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells underwent apoptosis in the presence of 3-bromopyruvate. This cell death was associated with the inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identified HDAC1 and HDAC3 as the targets for 3-bromopyruvate. 3-Bromopyruvate was transported into cells actively through the tumor suppressor SLC5A8, and the process was energized by an electrochemical Na+ gradient. Ectopic expression of the transporter in MCF7 cells led to apoptosis, and the mechanism involved the inhibition of HDAC1/HDAC3. Copyright (c) 2009 American Cancer Society.

Induction of Pancreatic Differentiation by Signals from Blood Vessels

NASA Astrophysics Data System (ADS)

Lammert, Eckhard; Cleaver, Ondine; Melton, Douglas

2001-10-01

Blood vessels supply developing organs with metabolic sustenance. Here, we demonstrate a role for blood vessels as a source of developmental signals during pancreatic organogenesis. In vitro experiments with embryonic mouse tissues demonstrate that blood vessel endothelium induces insulin expression in isolated endoderm. Removal of the dorsal aorta in Xenopus laevis embryos results in the failure of insulin expression in vivo. Furthermore, using transgenic mice, we show that ectopic vascularization in the posterior foregut leads to ectopic insulin expression and islet hyperplasia. These results indicate that vessels not only provide metabolic sustenance, but also provide inductive signals for organ development.

Enhanced epithelial to mesenchymal transition (EMT) and upregulated MYC in ectopic lesions contribute independently to endometriosis.

PubMed

Proestling, Katharina; Birner, Peter; Gamperl, Susanne; Nirtl, Nadine; Marton, Erika; Yerlikaya, Gülen; Wenzl, Rene; Streubel, Berthold; Husslein, Heinrich

2015-07-22

Epithelial to mesenchymal transition (EMT) is a process in which epithelial cells lose polarity and cell-to-cell contacts and acquire the migratory and invasive abilities of mesenchymal cells. These abilities are thought to be prerequisites for the establishment of endometriotic lesions. A hallmark of EMT is the functional loss of E-cadherin (CDH1) expression in epithelial cells. TWIST1, a transcription factor that represses E-cadherin transcription, is among the EMT inducers. SNAIL, a zinc-finger transcription factor, and its close relative SLUG have similar properties to TWIST1 and are thus also EMT inducers. MYC, which is upregulated by estrogens in the uterus by an estrogen response cis-acting element (ERE) in its promoter, is associated with proliferation in endometriosis. The role of EMT and proliferation in the pathogenesis of endometriosis was evaluated by analyzing TWIST1, CDH1 and MYC expression. CDH1, TWIST1, SNAIL and SLUG mRNA expression was analyzed by qRT-PCR from 47 controls and 74 patients with endometriosis. Approximately 42 ectopic and 62 eutopic endometrial tissues, of which 30 were matched samples, were collected during the same surgical procedure. We evaluated TWIST1 and MYC protein expression by immunohistochemistry (IHC) in the epithelial and stromal tissue of 69 eutopic and 90 ectopic endometrium samples, of which 49 matched samples were analyzed from the same patient. Concordant expression of TWIST1/SNAIL/SLUG and CDH1 but also of TWIST1 and MYC was analyzed. We found that TWIST1, SNAIL and SLUG are overexpressed (p < 0.001, p = 0.016 and p < 0.001) in endometriosis, while CDH1 expression was concordantly reduced in these samples (p < 0.001). Similar to TWIST1, the epithelial expression of MYC was also significantly enhanced in ectopic endometrium compared to eutopic tissues (p = 0.008). We found exclusive expression of either TWIST1 or MYC in the same samples (p = 0.003). Epithelial TWIST1 is overexpressed in endometriosis and may contribute to the formation of endometriotic lesions by inducing epithelial to mesenchymal transition, as CDH1 was reduced in ectopic lesions. We found exclusive expression of either TWIST1 or MYC in the same samples, indicating that EMT and proliferation contribute independently of each other to the formation of endometriotic lesions.

Ectopical expression of FABP4 gene can induce bovine muscle-derived stem cells adipogenesis.

PubMed

Zhang, Le; Zhao, Yanfang; Ning, Yue; Wang, Hongbao; Zan, Linsen

2017-01-08

Fatty acid binding protein 4 (FABP4) plays a key role in Fatty acid catabolism in mammals. Findings from our previous studies have indicated that FABP4 neither affect the differentiation of bovine preadipocytes nor does it change the expression of upstream genes. To investigate whether ectopically expressed FABP4 can induces Muscle-Derived Stem Cells (MDSCs) lipid synthesis and understand the regulatory mechanism behind it. In this study, adenoviruses infection is achieved to ectopically expressed FABP4 in bovine MDSCs, RNA-seq analyses at the very early stages of induction were performed to reveal gene expression level changes during MDSCs transdifferentiation. Results showed FABP4 can induce bovine Muscle-Derived Stem Cells transdifferentiation into adipocyte-like cells, 23 genes' expression levels changed after 24 h inducing although there is no significant change in cell phenotypes. Along with induction time, more differently expressed genes (256 genes changes after 48 h induction) were screened out. These genes should be at the downstream of signal pathways and be regulated by the 23 genes identified before. Our findings may provide a unique new model for studying the molecular control of cattle cross-talk between adipose and skeletal muscle. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Ectopic expression of the gastric inhibitory polypeptide receptor gene is a sufficient genetic event to induce benign adrenocortical tumor in a xenotransplantation model.

PubMed

Mazzuco, Tania L; Chabre, Olivier; Sturm, Nathalie; Feige, Jean-Jacques; Thomas, Michaël

2006-02-01

Aberrant expression of ectopic G protein-coupled receptors (GPCRs) in adrenal cortex tissue has been observed in several cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing's syndrome. Although there is clear clinical evidence for the implication of these ectopic receptors in abnormal regulation of cortisol production, whether this aberrant GPCR expression is the cause or the consequence of the development of an adrenal hyperplasia is still an open question. To answer it, we genetically engineered primary bovine adrenocortical cells to have them express the gastric inhibitory polypeptide receptor. After transplantation of these modified cells under the renal capsule of adrenalectomized immunodeficient mice, tissues formed had their functional and histological characteristics analyzed. We observed the formation of an enlarged and hyperproliferative adenomatous adrenocortical tissue that secreted cortisol in a gastric inhibitory polypeptide-dependent manner and induced a mild Cushing's syndrome with hyperglycemia. Moreover, we show that tumor development was ACTH independent. Thus, a single genetic event, inappropriate expression of a nonmutated GPCR gene, is sufficient to initiate the complete phenotypic alterations that ultimately lead to the formation of a benign adrenocortical tumor.

Lrp5/6 are required for cerebellar development and for suppressing TH expression in Purkinje cells via β-catenin.

PubMed

Huang, Ying; Zhang, Qiong; Song, Ning-Ning; Zhang, Lei; Sun, Yu-Ling; Hu, Ling; Chen, Jia-Ying; Zhu, Weidong; Li, Jue; Ding, Yu-Qiang

2016-01-15

The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons. We report here that the low-density lipoprotein receptors (Lrp) 5 and 6, Wnt co-receptors, are required for the development of the cerebellum and for suppressing ectopic TH expression in Purkinje cells. Simultaneous inactivation of Lrp 5 and 6 by Nestin-Cre results in defective lamination and foliation of the cerebellum during postnatal development. Surprisingly, TH is ectopically expressed by Purkinje cells, although they still keep its other neurochemical characteristics. These phenotypes are also observed in the cerebellum of GFAP-Cre;β-catenin(flox/flox) mice, and AAV2-Cre-mediated gene deletion leads to ectopic TH expression in Purkinje cells of β-catenin(flox/flox) mice as well. Our results revealed a new role of the canonical Lrp5/6-β-catenin pathway in regulating the morphogenesis of the cerebellum during postnatal development.

Restrictions in Cell Cycle Progression of Adult Vestibular Supporting Cells in Response to Ectopic Cyclin D1 Expression

PubMed Central

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H.; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27Kip1 and p21Cip1 expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells. PMID:22073316

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression.

PubMed

Loponen, Heidi; Ylikoski, Jukka; Albrecht, Jeffrey H; Pirvola, Ulla

2011-01-01

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.

Distal-less induces elemental color patterns in Junonia butterfly wings.

PubMed

Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Iwasaki, Mayo; Taira, Wataru; Adhikari, Kiran; Gurung, Raj; Otaki, Joji M

2016-01-01

The border ocellus, or eyespot, is a conspicuous color pattern element in butterfly wings. For two decades, it has been hypothesized that transcription factors such as Distal-less (Dll) are responsible for eyespot pattern development in butterfly wings, based on their expression in the prospective eyespots. In particular, it has been suggested that Dll is a determinant for eyespot size. However, functional evidence for this hypothesis has remained incomplete, due to technical difficulties. Here, we show that ectopically expressed Dll induces ectopic elemental color patterns in the adult wings of the blue pansy butterfly, Junonia orithya (Lepidoptera, Nymphalidae). Using baculovirus-mediated gene transfer, we misexpressed Dll protein fused with green fluorescent protein (GFP) in pupal wings, resulting in ectopic color patterns, but not the formation of intact eyespots. Induced changes included clusters of black and orange scales (a basic feature of eyespot patterns), black and gray scales, and inhibition of cover scale development. In contrast, ectopic expression of GFP alone did not induce any color pattern changes using the same baculovirus-mediated gene transfer system. These results suggest that Dll plays an instructive role in the development of color pattern elements in butterfly wings, although Dll alone may not be sufficient to induce a complete eyespot. This study thus experimentally supports the hypothesis of Dll function in eyespot development.

Senseless, a Zn finger transcription factor, is necessary and sufficient for sensory organ development in Drosophila

NASA Technical Reports Server (NTRS)

Nolo, R.; Abbott, L. A.; Bellen, H. J.

2000-01-01

The senseless (sens) gene is required for proper development of most cell types of the embryonic and adult peripheral nervous system (PNS) of Drosophila. Sens is a nuclear protein with four Zn fingers that is expressed and required in the sensory organ precursors (SOP) for proper proneural gene expression. Ectopic expression of Sens in many ectodermal cells causes induction of PNS external sensory organ formation and is able to recreate an ectopic proneural field. Hence, sens is both necessary and sufficient for PNS development. Our data indicate that proneural genes activate sens expression. Sens is then in turn required to further activate and maintain proneural gene expression. This feedback mechanism is essential for selective enhancement and maintenance of proneural gene expression in the SOPs.

Nuclear factor one B (NFIB) encodes a subtype-specific tumour suppressor in glioblastoma

PubMed Central

Stringer, Brett W.; Bunt, Jens; Day, Bryan W.; Barry, Guy; Jamieson, Paul R.; Ensbey, Kathleen S.; Bruce, Zara C.; Goasdoué, Kate; Vidal, Hélène; Charmsaz, Sara; Smith, Fiona M.; Cooper, Leanne T.; Piper, Michael

2016-01-01

Glioblastoma (GBM) is an essentially incurable and rapidly fatal cancer, with few markers predicting a favourable prognosis. Here we report that the transcription factor NFIB is associated with significantly improved survival in GBM. NFIB expression correlates inversely with astrocytoma grade and is lowest in mesenchymal GBM. Ectopic expression of NFIB in low-passage, patient-derived classical and mesenchymal subtype GBM cells inhibits tumourigenesis. Ectopic NFIB expression activated phospho-STAT3 signalling only in classical and mesenchymal GBM cells, suggesting a mechanism through which NFIB may exert its context-dependent tumour suppressor activity. Finally, NFIB expression can be induced in GBM cells by drug treatment with beneficial effects. PMID:27083054

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling

PubMed Central

Bechtold, Till E.; Saunders, Cheri; Decker, Rebekah S.; Um, Hyo-Bin; Cottingham, Naiga; Salhab, Imad; Kurio, Naito; Billings, Paul C.; Pacifici, Maurizio; Nah, Hyun-Duck; Koyama, Eiki

2016-01-01

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa’s articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis. PMID:26945615

Decreased expression of miR‑490‑3p in colorectal cancer predicts poor prognosis and promotes cell proliferation and invasion by targeting RAB14.

PubMed

Wang, Bo; Yin, Mujun; Cheng, Cheng; Jiang, Hongpeng; Jiang, Kewei; Shen, Zhanlong; Ye, Yingjiang; Wang, Shan

2018-06-19

Growing evidence indicates a potential role for miR‑490‑3p in tumorigenesis. However, its function in colorectal carcinoma (CRC) remains undefined. In this study, miR‑490‑3p was markedly downregulated in fifty colorectal cancer tissue samples compared with the corresponding adjacent non‑cancerous specimens, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of miR‑490‑3p were closely associated with tumor differentiation and distant metastasis. In addition, both Kaplan-Meier and multivariate analyses indicated CRC patients with elevated miR‑490‑3p amounts had prolonged overall survival. Overexpression of miR‑490‑3p markedly reduced proliferation, colony formation and invasion in CRC cells by enhancing apoptosis and promoting G2/M phase arrest. Furthermore, ectopic expression of miR‑490‑3p resulted in decreased expression of RAB14, which was directly targeted by miR‑490‑3p, as shown by the dual-luciferase reporter gene assay. Finally, in a nude mouse model, miR‑490‑3p overexpression significantly suppressed the growth of CRC cells. The above results indicated that miR‑490‑3p might constitute a prognostic indicator and a novel molecular target for miRNA-based CRC therapy.

Ectopic expression of homeobox gene NKX2-1 in diffuse large B-cell lymphoma is mediated by aberrant chromatin modifications.

PubMed

Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G; MacLeod, Roderick A F

2013-01-01

Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies.

The bHLH Repressor Deadpan Regulates the Self-renewal and Specification of Drosophila Larval Neural Stem Cells Independently of Notch

PubMed Central

Younger, Susan; Huang, Yaling; Lee, Tzumin

2012-01-01

Neural stem cells (NSCs) are able to self-renew while giving rise to neurons and glia that comprise a functional nervous system. However, how NSC self-renewal is maintained is not well understood. Using the Drosophila larval NSCs called neuroblasts (NBs) as a model, we demonstrate that the Hairy and Enhancer-of-Split (Hes) family protein Deadpan (Dpn) plays important roles in NB self-renewal and specification. The loss of Dpn leads to the premature loss of NBs and truncated NB lineages, a process likely mediated by the homeobox protein Prospero (Pros). Conversely, ectopic/over-expression of Dpn promotes ectopic self-renewing divisions and maintains NB self-renewal into adulthood. In type II NBs, which generate transit amplifying intermediate neural progenitors (INPs) like mammalian NSCs, the loss of Dpn results in ectopic expression of type I NB markers Asense (Ase) and Pros before these type II NBs are lost at early larval stages. Our results also show that knockdown of Notch leads to ectopic Ase expression in type II NBs and the premature loss of type II NBs. Significantly, dpn expression is unchanged in these transformed NBs. Furthermore, the loss of Dpn does not inhibit the over-proliferation of type II NBs and immature INPs caused by over-expression of activated Notch. Our data suggest that Dpn plays important roles in maintaining NB self-renewal and specification of type II NBs in larval brains and that Dpn and Notch function independently in regulating type II NB proliferation and specification. PMID:23056424

Indomethacin promotes apoptosis in gastric cancer cells through concomitant degradation of Survivin and Aurora B kinase proteins.

PubMed

Chiou, Shiun-Kwei; Hoa, Neil; Hodges, Amy; Ge, Lishen; Jadus, Martin R

2014-09-01

Regular usage of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with reduced incidence of a variety of cancers. The molecular mechanisms underlying these chemopreventive effects remain poorly understood. This current investigation showed that in gastric cancer cells: (1) Indomethacin treatment enhanced the degradation of chromosomal passenger proteins, Survivin and Aurora B kinase; (2) Indomethacin treatment down-regulated Aurora B kinase activity in a cell cycle-independent fashion; (3) siRNA knockdown of Survivin level promoted Aurora B kinase protein degradation, and vice versa; (4) ectopic overexpression of Survivin blocked reduction of Aurora B kinase level and activity by indomethacin treatment, and vice versa; (5) siRNA knockdown of Aurora B kinase level and AZD1152 inhibition of its activity induced apoptosis, and overexpression of Aurora B kinase inhibited indomethacin-induced apoptosis; (6) indomethacin treatment reduced Aurora B kinase level, coinciding with reduction of Survivin level and induction of apoptosis, in KATO III and HT-29 cells, and in mouse gastric mucosa. A role for Aurora B kinase function in NSAID-induced apoptosis was not previously explored. Thus this report provides better understanding of the molecular mechanisms underlying the anti-cancer effect of NSAIDs by elucidating a significant role for Aurora B kinase in indomethacin-induced apoptosis.

Interleukin-4 induces expression of eotaxin in endometriotic stromal cells.

PubMed

Ouyang, Zhuo; Osuga, Yutaka; Hirota, Yasushi; Hirata, Tetsuya; Yoshino, Osamu; Koga, Kaori; Yano, Tetsu; Taketani, Yuji

2010-06-01

To study the relationship between eotaxin and interleukin-4 (IL-4) in the pathophysiology of endometriosis. Comparative and laboratory study. University teaching hospital reproductive endocrinology and infertility practice. Ectopic endometrial tissues were collected from women with endometriosis. Ectopic endometrial stromal cells (ESCs) were isolated and cultured with IL-4. Ectopic endometriotic tissues were immunostained for eotaxin and IL-4. Gene expression of eotaxin was determined by standard and real-time reverse-transcriptase polymerase chain reaction. Secretion of eotaxin from ESC was measured using specific ELISA. The immunostained sections were examined. Interleukin-4 (IL-4) increased mRNA expression and protein secretion of eotaxin from ESC in a dose-dependent manner. Immunohistochemical analysis showed that eotaxin-positive cells colocalized with IL-4-positive cells and accumulated around the blood vessels in the stroma of endometriotic tissue. IL-4 induces eotaxin in ESCs, which might promote angiogenesis and the subsequent development of endometriosis. Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Growth enhancement in transgenic tilapia by ectopic expression of tilapia growth hormone.

PubMed

Martínez, R; Estrada, M P; Berlanga, J; Guillén, I; Hernández, O; Cabrera, E; Pimentel, R; Morales, R; Herrera, F; Morales, A; Piña, J C; Abad, Z; Sánchez, V; Melamed, P; Lleonart, R; de la Fuente, J

1996-03-01

The generation of transgenic fish with the transfer of growth hormone (GH) genes has opened new possibilities for the manipulation of growth in economically important fish species. The tilapia growth hormone (tiGH) cDNA was linked to the human cytomegalovirus (CMV) enhancer-promoter and used to generate transgenic tilapia by microinjection into one-cell embryos. Five transgenic tilapia were obtained from 40 injected embryos. A transgenic animal containing one copy of the transgene per cell was selected to establish a transgenic line. The transgene was stably transmitted to F1 and F2 generations in a Mendelian fashion. Ectopic, low-level expression of tiGH was detected in gonad and muscle cells of F1 transgenic tilapia by immunohystochemical analysis of tissue sections. Nine-month-old transgenic F1 progeny were 82% larger than nontransgenic fish at p = .001. These results showed that low-level ectopic expression of tiGH resulted in a growth acceleration in transgenic tilapia. Tilapia GH gene transfer is an alternative for growth acceleration in tilapia.

p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax

PubMed Central

Kawata, Sanae; Ariumi, Yasuo; Shimotohno, Kunitada

2003-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21Waf1/Cip1/Sdi1, resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-κB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms. PMID:12805427

The homeodomain transcription factor Cdx1 does not behave as an oncogene in normal mouse intestine.

PubMed

Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

2008-01-01

The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

PubMed

Caserta, R; Souza-Neto, R R; Takita, M A; Lindow, S E; De Souza, A A

2017-11-01

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Pokemon silencing leads to Bim-mediated anoikis of human hepatoma cell QGY7703.

PubMed

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

PubMed Central

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer. PMID:22754333

The cleft lip and palate defects in Dancer mutant mice result from gain of function of the Tbx10 gene

PubMed Central

Bush, Jeffrey O.; Lan, Yu; Jiang, Rulang

2004-01-01

Cleft lip and palate (CL/P) is a common disfiguring birth defect with complex, poorly understood etiology. Mice carrying a spontaneous mutation, Dancer (Dc), exhibit CL/P in homozygotes and show significantly increased susceptibility to CL/P in heterozygotes [Deol, M. S. & Lane, P. W. (1966) J. Embryol. Exp. Morphol. 16, 543–558 and Trasler, D. G., Kemp, D. & Trasler, T. A. (1984) Teratology 29, 101–104], providing an animal model for understanding the molecular pathogenesis of CL/P. We genetically mapped Dc to within a 1-cM region near the centromere of chromosome 19. In situ hybridization analysis showed that one positional candidate gene, Tbx10, is ectopically expressed in Dc mutant embryos. Positional cloning of the Dc locus revealed an insertion of a 3.3-kb sequence containing the 5′ region of the p23 gene into the first intron of Tbx10, which causes ectopic expression of a p23-Tbx10 chimeric transcript encoding a protein product identical to a normal variant of the Tbx10 protein. Furthermore, we show that ectopic expression of Tbx10 in transgenic mice recapitulates the Dc mutant phenotype, indicating that CL/Pin Dc mutant mice results from the p23 insertion-induced ectopic Tbx10 expression. These results identify gain of function of a T-box transcription factor gene as a mechanism underlying CL/P pathogenesis. PMID:15118109

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

PubMed

Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z; Wickrema, Amittha; Yang, Jing; Ji, Peng

2016-03-07

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

Nuclear condensation during mouse erythropoiesis requires caspase-3-mediated nuclear opening

PubMed Central

Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z.; Wickrema, Amittha; Yang, Jing; Ji, Peng

2016-01-01

SUMMARY Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step towards chromatin condensation during erythropoiesis in mice. PMID:26954545

An endogenous and ectopic expression of metabotropic glutamate receptor 8 (mGluR8) inhibits proliferation and increases chemosensitivity of human neuroblastoma and glioma cells.

PubMed

Jantas, Danuta; Grygier, Beata; Gołda, Sławomir; Chwastek, Jakub; Zatorska, Justyna; Tertil, Magdalena

2018-06-06

The present study aimed to determine the role of metabotropic glutamate receptor 8 (mGluR8) in tumor biology. Using various molecular approaches (RNAi or GRM8 cDNA), cell clones with downregulated (human neuroblastoma SH-SY5Y and human glioma LN229) or overexpressed (human glioma U87-MG and LN18 cell lines) mGluR8 were generated. Next, comparative studies on cell proliferation and migration rates, induction of apoptosis and chemosensitivity were performed among these clones. The mGluR8-downregulated SH-SY5Y clones proliferated faster and were more resistant to cytotoxic action of staurosporine, doxorubicin, irinotecan and cisplatin when compared to control cells. Moreover, these clones were characterized by a lower activity of caspases, calpains and some kinases (GSK-3β, Akt and JNK). The mGluR8-downregulated LN229 clones migrated faster and were less prone to cell-damaging effect of staurosporine and irinotecan when compared with relevant control cells. In contrast, in GRM8-overexpressing U87-MG and LN18 clones, a decreased cell proliferation, increased apoptosis and elevated vulnerability to some cytotoxic agents were found. Altogether, our in vitro data for the first time evidenced a tumor suppressor and chemosensitizing role of mGluR8. Copyright © 2018 Elsevier B.V. All rights reserved.

Palmitate Attenuates Osteoblast Differentiation of Fetal Rat Calvarial Cells

PubMed Central

Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.; Adamo, Martin L.

2014-01-01

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl Co-A carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPAR gamma in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. PMID:24955854

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells.

PubMed

Yeh, Lee-Chuan C; Ford, Jeffery J; Lee, John C; Adamo, Martin L

2014-07-18

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. Copyright © 2014 Elsevier Inc. All rights reserved.

The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome.

PubMed Central

de Keyzer, Y; Lenne, F; Auzan, C; Jégou, S; René, P; Vaudry, H; Kuhn, J M; Luton, J P; Clauser, E; Bertagna, X

1996-01-01

Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome. PMID:8636444

The pituitary V3 vasopressin receptor and the corticotroph phenotype in ectopic ACTH syndrome.

PubMed

de Keyzer, Y; Lenne, F; Auzan, C; Jégou, S; René, P; Vaudry, H; Kuhn, J M; Luton, J P; Clauser, E; Bertagna, X

1996-03-01

Ectopic ACTH secretion occurs in highly differentiated and rather indolent tumors like bronchial carcinoids or, in contrast, in various types of aggressive and poorly differentiated neuroendocrine tumors. We explored this phenomenon using the recently cloned human pituitary V3 vasopressin receptor as an alternate molecular marker of the corticotroph phenotype. Expression of V3 receptor, corticotrophin releasing hormone (CRH) receptor, and proopiomelanocortin (POMC) genes was examined in tumors of pituitary and nonpituitary origin. A comparative RT-PCR approach revealed signals for both V3 receptor and CHR receptor mRNAs in 17 of 18 ACTH-secreting pituitary adenomas, and 6 of 6 normal pituitaries; in six growth hormone- or prolactin-secreting adenomas, a very faint V3 receptor signal was observed in three cases, and CRH receptor signal was undetected in all. Six of eight bronchial carcinoids responsible for the ectopic ACTH syndrome had both POMC and V3 receptor signals as high as those in ACTH-secreting pituitary adenomas; in contrast, no POMC signal and only a very faint V3 receptor signal were detected in six of eight nonsecreting bronchial carcinoids. Northern blot analysis showed V3 receptor mRNA of identical size in ACTH-secreting bronchial carcinoids and pituitary tumors. Other types of nonpituitary tumors responsible for ectopic ACTH syndrome presented much lower levels of both POMC and V3 receptor gene expression than those found in ACTH-secreting bronchial carcinoids. In contrast with the V3 receptor, CRH receptor mRNA was detected in the majority of neuroendocrine tumors irrespective of their POMC status. These results show that expression of the V3 receptor gene participates in the corticotroph phenotype. Its striking association with ACTH-secreting bronchial carcinoids defines a subset of nonpituitary tumors in which ectopic POMC gene expression is but one aspect of a wider process of corticotroph cell differentiation, and opens new possibilities of pharmacological investigations and even manipulations of this peculiar ACTH hypersecretory syndrome.

Ectopic Expression of Homeobox Gene NKX2-1 in Diffuse Large B-Cell Lymphoma Is Mediated by Aberrant Chromatin Modifications

PubMed Central

Nagel, Stefan; Ehrentraut, Stefan; Tomasch, Jürgen; Quentmeier, Hilmar; Meyer, Corinna; Kaufmann, Maren; Drexler, Hans G.; MacLeod, Roderick A. F.

2013-01-01

Homeobox genes encode transcription factors ubiquitously involved in basic developmental processes, deregulation of which promotes cell transformation in multiple cancers including hematopoietic malignancies. In particular, NKL-family homeobox genes TLX1, TLX3 and NKX2-5 are ectopically activated by chromosomal rearrangements in T-cell neoplasias. Here, using transcriptional microarray profiling and RQ-PCR we identified ectopic expression of NKL-family member NKX2-1, in a diffuse large B-cell lymphoma (DLBCL) cell line SU-DHL-5. Moreover, in silico analysis demonstrated NKX2-1 overexpression in 5% of examined DLBCL patient samples. NKX2-1 is physiologically expressed in lung and thyroid tissues where it regulates differentiation. Chromosomal and genomic analyses excluded rearrangements at the NKX2-1 locus in SU-DHL-5, implying alternative activation. Comparative expression profiling implicated several candidate genes in NKX2-1 regulation, variously encoding transcription factors, chromatin modifiers and signaling components. Accordingly, siRNA-mediated knockdown and overexpression studies confirmed involvement of transcription factor HEY1, histone methyltransferase MLL and ubiquitinated histone H2B in NKX2-1 deregulation. Chromosomal aberrations targeting MLL at 11q23 and the histone gene cluster HIST1 at 6p22 which we observed in SU-DHL-5 may, therefore, represent fundamental mutations mediating an aberrant chromatin structure at NKX2-1. Taken together, we identified ectopic expression of NKX2-1 in DLBCL cells, representing the central player in an oncogenic regulative network compromising B-cell differentiation. Thus, our data extend the paradigm of NKL homeobox gene deregulation in lymphoid malignancies. PMID:23637834

Reverse-phase protein array profiling of oropharyngeal cancer and significance of PIK3CA mutations in HPV-associated head and neck cancer.

PubMed

Sewell, Andrew; Brown, Brandee; Biktasova, Asel; Mills, Gordon B; Lu, Yiling; Tyson, Darren R; Issaeva, Natalia; Yarbrough, Wendell G

2014-05-01

Human papilloma virus (HPV)-associated (HPV+) oropharyngeal squamous cell carcinomas (OPSCC) have different molecular and biologic characteristics and clinical behavior compared with HPV-negative (HPV-) OPSCC. PIK3CA mutations are more common in HPV(+) OPSCC. To define molecular differences and tumor subsets, protein expression and phosphorylation were compared between HPV(+) and HPV(-) OPSCC and between tumors with and without PIK3CA mutations. Expression of 137 total and phosphorylated proteins was evaluated by reverse-phase protein array in 29 HPV(+) and 13 HPV(-) prospectively collected OPSCCs. Forty-seven OPSCCs were tested for hotspot-activating mutations in PIK3CA and AKT. Activation of PIK3CA downstream targets and sensitivity to pathway inhibitors were determined in HPV(+) head and neck cancer cells overexpressing wild-type or mutant PIK3CA. Analyses revealed 41 differentially expressed proteins between HPV(+) and HPV(-) OPSCC categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. All queried DNA repair proteins were significantly upregulated in HPV(+) samples. A total of 8 of 33 HPV(+) and 0 of 14 HPV(-) tumors contained activating PIK3CA mutations. Despite all activating PIK3CA mutations occurring in HPV(+) samples, HPV(+) tumors had lower mean levels of activated AKT and downstream AKT target phosphorylation. Ectopic expression of mutant PIK3CA in HPV(+) cells increased mTOR, but not AKT activity. HPV E6/E7 overexpression inhibited AKT phosphorylation in HPV-negative cells. Mutant PIK3CA overexpressing cells were more sensitive to a dual PI3K/mTOR inhibitor compared with an AKT inhibitor. Protein expression analyses suggest that HPV(+) and HPV(-) OPSCC differentially activate DNA repair, cell cycle, apoptosis, PI3K/AKT/mTOR, and receptor kinase pathways. PIK3CA mutations are more common in HPV(+) OPSCC and are associated with activation of mTOR, but not AKT. These data suggest that inhibitors for mTOR may have activity against HPV(+) PIK3CA mutant oropharyngeal cancers. ©2014 AACR.

Ectopic norrin induces growth of ocular capillaries and restores normal retinal angiogenesis in Norrie disease mutant mice.

PubMed

Ohlmann, Andreas; Scholz, Michael; Goldwich, Andreas; Chauhan, Bharesh K; Hudl, Kristiane; Ohlmann, Anne V; Zrenner, Eberhart; Berger, Wolfgang; Cvekl, Ales; Seeliger, Mathias W; Tamm, Ernst R

2005-02-16

Norrie disease is an X-linked retinal dysplasia that presents with congenital blindness, sensorineural deafness, and mental retardation. Norrin, the protein product of the Norrie disease gene (NDP), is a secreted protein of unknown biochemical function. Norrie disease (Ndp(y/-)) mutant mice that are deficient in norrin develop blindness, show a distinct failure in retinal angiogenesis, and completely lack the deep capillary layers of the retina. We show here that the transgenic expression of ectopic norrin under control of a lens-specific promoter restores the formation of a normal retinal vascular network in Ndp(y/-) mutant mice. The improvement in structure correlates with restoration of neuronal function in the retina. In addition, lenses of transgenic mice with ectopic expression of norrin show significantly more capillaries in the hyaloid vasculature that surrounds the lens during development. In vitro, lenses of transgenic mice in coculture with microvascular endothelial cells induce proliferation of the cells. Transgenic mice with ectopic expression of norrin show more bromodeoxyuridine-labeled retinal progenitor cells at embryonic day 14.5 and thicker retinas at postnatal life than wild-type littermates, indicating a putative direct neurotrophic effect of norrin. These data provide direct evidence that norrin induces growth of ocular capillaries and that pharmacologic modulation of norrin might be used for treatment of the vascular abnormalities associated with Norrie disease or other vascular disorders of the retina.

Inhibition of BET Bromodomain Targets Genetically Diverse Glioblastoma

PubMed Central

Cheng, Zhixiang; Gong, Yuanying; Ma, Yufang; Lu, Kaihua; Lu, Xiang; Pierce, Larry A.; Thompson, Reid C.; Muller, Susanne; Knapp, Stefan; Wang, Jialiang

2014-01-01

Purpose Glioblastoma is refractory to conventional therapies. The bromodomain and extraterminal domain (BET) proteins are epigenetic readers that selectively bind to acetylated lysine residues on histone tails. These proteins recently emerged as important therapeutic targets in NUT midline carcinoma and several types of hematopoietic cancers. In this study, the therapeutic potential of a novel BET bromodomain inhibitor, JQ1, was assessed in a panel of genetically heterogeneous glioblastoma samples. Experimental Design The antineoplastic effects of JQ1 were shown using ex vivo cultures derived from primary glioblastoma xenograft lines and surgical specimens of different genetic background. The in vivo efficacy was assessed in orthotopic glioblastoma tumors. Results We showed that JQ1 induced marked G1 cell-cycle arrest and apoptosis, which was phenocopied by knockdown of individual BET family members. JQ1 treatment resulted in significant changes in expression of genes that play important roles in glioblastoma such as c-Myc, p21CIP1/WAF1, hTERT, Bcl-2, and Bcl-xL. Unlike the observations in some hematopoietic cancer cell lines, exogenous c-Myc did not significantly protect glioblastoma cells against JQ1. In contrast, ectopically expressed Bcl-xL partially rescued cells from JQ1-induced apoptosis, and knockdown of p21CIP1/WAF1 attenuated JQ1-induced cell-cycle arrest. Cells genetically engineered for Akt hyperactivation or p53/Rb inactivation did not compromise JQ1 efficacy, suggesting that these frequently mutated signaling pathways may not confer resistance to JQ1. Furthermore, JQ1 significantly repressed growth of orthotopic glioblastoma tumors. Conclusion Our results suggest potentially broad therapeutic use of BET bromodomain inhibitors for treating genetically diverse glioblastoma tumors. PMID:23403638

Epstein-Barr virus nuclear antigen 3C targets p53 and modulates its transcriptional and apoptotic activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi Fuming; Saha, Abhik; Murakami, Masanao

The p53 tumor suppressor gene is one of the most commonly mutated genes in human cancers and the corresponding encoded protein induces apoptosis or cell-cycle arrest at the G1/S checkpoint in response to DNA damage. To date, previous studies have shown that antigens encoded by human tumor viruses such as SV40 large T antigen, adenovirus E1A and HPV E6 interact with p53 and disrupt its functional activity. In a similar fashion, we now show that EBNA3C, one of the EBV latent antigens essential for the B-cell immortalization in vitro, interacts directly with p53. Additionally, we mapped the interaction of EBNA3Cmore » with p53 to the C-terminal DNA-binding and the tetramerization domain of p53, and the region of EBNA3C responsible for binding to p53 was mapped to the N-terminal domain of EBNA3C (residues 130-190), previously shown to interact with a number of important cell-cycle components, specifically SCF{sup Skp2}, cyclin A, and cMyc. Furthermore, we demonstrate that EBNA3C substantially represses the transcriptional activity of p53 in luciferase based reporter assays, and rescues apoptosis induced by ectopic p53 expression in SAOS-2 (p53{sup -/-}) cells. Interestingly, we also show that the DNA-binding ability of p53 is diminished in the presence of EBNA3C. Thus, the interaction between the p53 and EBNA3C provides new insights into the mechanism(s) by which the EBNA3C oncoprotein can alter cellular gene expression in EBV associated human cancers.« less

FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

PubMed

Mak, Victor C Y; Wong, Oscar G W; Siu, Michelle K Y; Wong, Esther S Y; Ng, Wai-Yan; Wong, Richard W C; Chan, Ka-Kui; Ngan, Hextan Y S; Cheung, Annie N Y

2015-07-01

Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Mutations of the Mitochondrial Holocytochrome c–Type Synthase in X-Linked Dominant Microphthalmia with Linear Skin Defects Syndrome

PubMed Central

Wimplinger, Isabella; Morleo, Manuela; Rosenberger, Georg; Iaconis, Daniela; Orth, Ulrike; Meinecke, Peter; Lerer, Israela; Ballabio, Andrea; Gal, Andreas; Franco, Brunella; Kutsche, Kerstin

2006-01-01

The microphthalmia with linear skin defects syndrome (MLS, or MIDAS) is an X-linked dominant male-lethal disorder almost invariably associated with segmental monosomy of the Xp22 region. In two female patients, from two families, with MLS and a normal karyotype, we identified heterozygous de novo point mutations—a missense mutation (p.R217C) and a nonsense mutation (p.R197X)—in the HCCS gene. HCCS encodes the mitochondrial holocytochrome c–type synthase that functions as heme lyase by covalently adding the prosthetic heme group to both apocytochrome c and c1. We investigated a third family, displaying phenotypic variability, in which the mother and two of her daughters carry an 8.6-kb submicroscopic deletion encompassing part of the HCCS gene. Functional analysis demonstrates that both mutant proteins (R217C and Δ197–268) were unable to complement a Saccharomyces cerevisiae mutant deficient for the HCCS orthologue Cyc3p, in contrast to wild-type HCCS. Moreover, ectopically expressed HCCS wild-type and the R217C mutant protein are targeted to mitochondria in CHO-K1 cells, whereas the C-terminal–truncated Δ197–268 mutant failed to be sorted to mitochondria. Cytochrome c, the final product of holocytochrome c–type synthase activity, is implicated in both oxidative phosphorylation (OXPHOS) and apoptosis. We hypothesize that the inability of HCCS-deficient cells to undergo cytochrome c–mediated apoptosis may push cell death toward necrosis that gives rise to severe deterioration of the affected tissues. In summary, we suggest that disturbance of both OXPHOS and the balance between apoptosis and necrosis, as well as the X-inactivation pattern, may contribute to the variable phenotype observed in patients with MLS. PMID:17033964

Peptidyl-prolyl cis/trans isomerase Pin1 regulates withaferin A-mediated cell cycle arrest in human breast cancer cells.

PubMed

Samanta, Suman K; Lee, Joomin; Hahm, Eun-Ryeong; Singh, Shivendra V

2018-07-01

We have reported previously that withaferin A (WA) prevents breast cancer development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice, but the mechanism is not fully understood. Unbiased proteomics of the mammary tumors from control- and WA-treated MMTV-neu mice revealed downregulation of peptidyl-prolyl cis/trans isomerase (Pin1) protein by WA administration. The present study extends these findings to elucidate the role of Pin1 in cancer chemopreventive mechanisms of WA. The mammary tumor level of Pin1 protein was lower by about 55% in WA-treated rats exposed to N-methyl-N-nitrosourea, compared to control. Exposure of MCF-7 and SK-BR-3 human breast cancer cells to WA resulted in downregulation of Pin1 protein. Ectopic expression of Pin1 attenuated G 2 and/or mitotic arrest resulting from WA treatment in both MCF-7 and SK-BR-3 cells. WA-induced apoptosis was increased by Pin1 overexpression in MCF-7 cells but not in the SK-BR-3 cell line. In addition, molecular docking followed by mass spectrometry indicated covalent interaction of WA with cysteine 113 of Pin1. Overexpression of Pin1 C113A mutant failed to attenuate WA-induced mitotic arrest or apoptosis in the MCF-7 cells. Furthermore, antibody array revealed upregulation of proapoptotic insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, IGFBP-4, IGFBP-5, and IGFBP-6, in Pin1 overexpressing MCF-7 cells following WA treatment when compared to empty vector transfected control cells. These data support a crucial role of the Pin1 for mitotic arrest and apoptosis signaling by WA at least in the MCF-7 cells. © 2018 Wiley Periodicals, Inc.

Association of nbl gene expression and glucocorticoid-induced apoptosis in mouse thymus in vivo.

PubMed

Naora, H; Nishida, T; Shindo, Y; Adachi, M; Naora, H

1995-05-01

A gene of unknown biological function, nbl, was originally isolated by virtue of its abundance in a Namalwa Burkitt Lymphoma cDNA library. nbl expression was initially found to be higher in tissues which exhibited internucleosomal DNA cleavage characteristic of apoptosis, than in tissues which did not exhibit a 'DNA ladder'. nbl expression was therefore examined in mouse thymus in vivo, in which apoptosis is induced by the glucocorticoid, dexamethasone. nbl expression was markedly enhanced by dexamethasone treatment and then sharply decreased prior to the occurrence of maximal 'DNA ladder' formation. In contrast, expression of myc, which is believed to be involved in apoptosis in other cell systems, declined as thymic apoptosis increased. Thymic apoptosis was blocked by the transcriptional inhibitor actinomycin D, if administered when nbl expression was enhanced, but not before or after the peak of nbl expression. These results suggest that nbl expression is associated with thymic apoptosis.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Hepatic leukemia factor promotes resistance to cell death: Implications for therapeutics and chronotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waters, Katrina M.; Sontag, Ryan L.; Weber, Thomas J., E-mail: Thomas.Weber@pnl.gov

Physiological variation related to circadian rhythms and aberrant gene expression patterns are believed to modulate therapeutic efficacy, but the precise molecular determinants remain unclear. Here we examine the regulation of cell death by hepatic leukemia factor (HLF), which is an output regulator of circadian rhythms and is aberrantly expressed in human cancers, using an ectopic expression strategy in JB6 mouse epidermal cells and human keratinocytes. Ectopic HLF expression inhibited cell death in both JB6 cells and human keratinocytes, as induced by serum-starvation, tumor necrosis factor alpha and ionizing radiation. Microarray analysis indicates that HLF regulates a complex multi-gene transcriptional programmore » encompassing upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes, and many additional changes that are consistent with an anti-death program. Collectively, our results demonstrate that ectopic expression of HLF, an established transcription factor that cycles with circadian rhythms, can recapitulate many features associated with circadian-dependent physiological variation. - Highlights: ► Circadian-dependent physiological variation impacts therapeutic efficacy. ► Hepatic leukemia factor inhibits cell death and is a candidate circadian factor. ► Hepatic leukemia factor anti-death program is conserved in murine and human cells. ► Transcriptomics indicates the anti-death program results from a systems response.« less

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis

PubMed Central

Jiang, Xiu Xiu; Fei, Xiang Wei; Zhao, Li; Ye, Xiao Lei; Xin, Liao Bin; Qu, Yang; Xu, Kai Hong; Wu, Rui Jin; Lin, Jun

2015-01-01

Aquaporin 5 (AQP5) participates in the migration of endometrial cells. Elucidation of the molecular mechanisms associated with AQP5-mediated, migration of endometrial cells may contribute to a better understanding of endometriosis. Our objectives included identifying the estrogen-response element (ERE) in the promoter region of the AQP5 gene, and, investigating the effects of AQP5 on ectopic implantation of endometrial cells. Luciferase reporter assays and electrophoretic mobility shift assay (EMSA) identified the ERE-like motif in the promoter region of the AQP5 gene. After blocking and up-regulating estradiol (E2) levels, we analysed the expression of AQP5 in endometrial stromal (ES) cells. After blocking E2 /or phosphatidylinositol 3 kinase(PI3K), we analysed the role of AQP5 in signaling pathways. We constructed an AQP5, shRNA, lentiviral vector to knock out the AQP5 gene in ES cells. After knock-out of the AQP5 gene, we studied the role of AQP5 in cell invasion, proliferation, and the formation of ectopic endometrial implants in female mice. We identified an estrogen-response element in the promoter region of the AQP5 gene. Estradiol (E2) increased AQP5 expression in a dose-dependent fashion, that was blocked by ICI182,780(an estrogen receptor inhibitor). E2 activated PI3K /protein kinase B(AKT) pathway (PI3K/AKT), that, in turn, increased AQP5 expression. LY294002(PI3K inhibitor) attenuated estrogen-enhanced, AQP5 expression. Knock-out of the AQP5 gene with AQP5 shRNA lentiviral vector significantly inhibited E2-enhanced invasion, proliferation of ES cells and formation of ectopic implants. Estrogen induces AQP5 expression by activating ERE in the promoter region of the AQP5gene, activates the PI3K/AKT pathway, and, promotes endometrial cell invasion and proliferation. These results provide new insights into some of the mechanisms that may underpin the development of deposits of ectopic endometrium. PMID:26679484

Wnt/β-Catenin Regulates the Activity of Epiprofin/Sp6, SHH, FGF, and BMP to Coordinate the Stages of Odontogenesis

PubMed Central

Aurrekoetxea, Maitane; Irastorza, Igor; García-Gallastegui, Patricia; Jiménez-Rojo, Lucia; Nakamura, Takashi; Yamada, Yoshihiko; Ibarretxe, Gaskon; Unda, Fernando J.

2016-01-01

Background: We used an in vitro tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO), a specific inhibitor of GSK-3 activity. Results: Overactivating the Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers Sonic Hedgehog (Shh), Epiprofin (Epfn), and Fibroblast growth factor8 (Fgf8), which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis, Fibroblast growth factor4 (Fgf4), Fibroblast growth factor10 (Fgf10), Muscle segment homeobox 1 (Msx-1), Bone Morphogenetic protein 4 (Bmp4), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1) were overexpressed in first molars cultured with BIO. Conversely, the expression levels of Wingless integration site 10b (Wnt-10b) and Shh were reduced. Additionally, the odontoblast differentiation markers Nestin and Epfn showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated Epfn expression during dental morphogenesis. Conclusions: We suggest the presence of a positive feedback loop wherein Epfn and β-catenin activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp, and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts. PMID:27066482

Transdifferentiation mediated tumor suppression by the endoplasmic reticulum stress sensor IRE-1 in C. elegans

PubMed Central

Levi-Ferber, Mor; Gian, Hai; Dudkevich, Reut; Henis-Korenblit, Sivan

2015-01-01

Deciphering effective ways to suppress tumor progression and to overcome acquired apoptosis resistance of tumor cells are major challenges in the tumor therapy field. We propose a new concept by which tumor progression can be suppressed by manipulating tumor cell identity. In this study, we examined the effect of ER stress on apoptosis resistant tumorous cells in a Caenorhabditis elegans germline tumor model. We discovered that ER stress suppressed the progression of the lethal germline tumor by activating the ER stress sensor IRE-1. This suppression was associated with the induction of germ cell transdifferentiation into ectopic somatic cells. Strikingly, transdifferentiation of the tumorous germ cells restored their ability to execute apoptosis and enabled their subsequent removal from the gonad. Our results indicate that tumor cell transdifferentiation has the potential to combat cancer and overcome the escape of tumor cells from the cell death machinery. DOI: http://dx.doi.org/10.7554/eLife.08005.001 PMID:26192965

The Homeodomain Transcription Factor Cdx1 Does Not Behave as an Oncogene in Normal Mouse Intestine1

PubMed Central

Crissey, Mary Ann S; Guo, Rong-Jun; Fogt, Franz; Li, Hong; Katz, Jonathan P; Silberg, Debra G; Suh, Eun Ran; Lynch, John P

2008-01-01

The Caudal-related homeobox genes Cdx1 and Cdx2 are intestine-specific transcription factors that regulate differentiation of intestinal cell types. Previously, we have shown Cdx1 to be antiproliferative and to promote cell differentiation. However, other studies have suggested that Cdx1 may be an oncogene. To test for oncogenic behavior, we used the murine villin promoter to ectopically express Cdx1 in the small intestinal villi and colonic surface epithelium. No changes in intestinal architecture, cell differentiation, or lineage selection were observed with expression of the transgene. Classic oncogenes enhance proliferation and induce tumors when ectopically expressed. However, the Cdx1 transgene neither altered intestinal proliferation nor induced spontaneous intestinal tumors. In a murine model for colitis-associated cancer, the Cdx1 transgene decreased, rather than increased, the number of adenomas that developed. In the polyps, the expression of the endogenous and the transgenic Cdx1 proteins was largely absent, whereas endogenous Villin expression was retained. This suggests that transgene silencing was specific and not due to a general Villin inactivation. In conclusion, neither the ectopic expression of Cdx1 was associated with changes in intestinal cell proliferation or differentiation nor was there increased intestinal cancer susceptibility. Our results therefore suggest that Cdx1 is not an oncogene in normal intestinal epithelium. PMID:18231635

Ectopic Fgf signaling induces the intercalary response in developing chicken limb buds.

PubMed

Makanae, Aki; Satoh, Akira

2018-01-01

Intercalary pattern formation is an important regulatory step in amphibian limb regeneration. Amphibian limb regeneration is composed of multiple steps, including wounding, blastema formation, and intercalary pattern formation. Attempts have been made to transfer insights from regeneration-competent animals to regeneration-incompetent animalsat each step in the regeneration process. In the present study, we focused on the intercalary mechanism in chick limb buds. In amphibian limb regeneration, a proximodistal axis is organized as soon as a regenerating blastema is induced. Intermediate structures are subsequently induced (intercalated) between the established proximal and distal identities. Intercalary tissues are derived from proximal tissues. Fgf signaling mediates the intercalary response in amphibian limb regeneration. We attempted to transfer insights into intercalary regeneration from amphibian models to the chick limb bud. The zeugopodial part was dissected out, and the distal and proximal parts were conjunct at st. 24. Delivering ectopic Fgf2 + Fgf8 between the distal and proximal parts resulted in induction of zeugopodial elements. Examination of HoxA11 expression, apoptosis, and cell proliferation provides insights to compare with those in the intercalary mechanism of amphibian limb regeneration. Furthermore, the cellular contribution was investigated in both the chicken intercalary response and that of axolotl limb regeneration. We developed new insights into cellular contribution in amphibian intercalary regeneration, and found consistency between axolotl and chicken intercalary responses. Our findings demonstrate that the same principal of limb regeneration functions between regeneration-competent and -incompetent animals. In this context, we propose the feasibility of the induction of the regeneration response in amniotes.

Methylation of Septin9 mediated by DNMT3a enhances hepatic stellate cells activation and liver fibrogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Yuting, E-mail: wuyuting1302@sina.com; Bu, Fan

Liver fibrosis, resulting from chronic and persistent injury to the liver, is a worldwide health problem. Advanced liver fibrosis results in cirrhosis, liver failure and even hepatocellular cancer (HCC), often eventually requiring liver transplantation, poses a huge health burden on the global community. However, the specific pathogenesis of liver fibrosis remains not fully understood. Numerous basic and clinical studies have provided evidence that epigenetic modifications, especially DNA methylation, might contribute to the activation of hepatic stellate cells (HSCs), the pivotal cell type responsible for the fibrous scar in liver. Here, reduced representation bisulfite sequencing (RRBS) and bisulfite pyrosequencing PCR (BSP)more » analysis identified hypermethylation status of Septin9 (Sept9) gene in liver fibrogenesis. Sept9 protein was dramatically decreased in livers of CCl4-treated mice and immortalized HSC-T6 cells exposed to TGF-β1. Nevertheless, the suppression of Sept9 could be blocked by DNMT3a-siRNA and DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5-azadC). Overexpressed Sept9 attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and Col1a1, accompanied by up-regulation of cell apoptosis-related proteins. Conversely, RNAi-mediated silencing of Sept9 enhanced accumulation of extracellular matrix. These observations suggested that Sept9 contributed to alleviate liver fibrosis might partially through promoting activated HSCs apoptosis and this anti-fibrogenesis effect might be blocked by DNMT-3a mediated methylation of Sept9. Therefore, pharmacological agents that inhibit Sept9 methylation and increase its expression could be considered as valuable treatments for liver fibrosis. - Highlights: • This is the first report of Sept9 methylation and function in liver fibrosis. • Ectopic expression of Sept9 could block the liver fibrogenesis. • DNMT3a might be responsible for the suppression of Sept9 in liver fibrosis.« less

MiR-128b is down-regulated in gastric cancer and negatively regulates tumour cell viability by targeting PDK1/Akt/NF-κB axis.

PubMed

Zhang, Ling; Lei, Jun; Fang, Zi-Ling; Xiong, Jian-Ping

2016-03-01

Gastric cancer (GC) is the fourth most prevalent type of cancer worldwide, which is usually caused by the interaction between environmental and genetic factors, or epigenetic aspects. Referring to the non-coding RNAs, miR-128b has been reported to be associated with many tumour cases, and exerts distinct functions in different types of cancers. However, the function of miR-128b in GC onset and progression largely remains unknown. In the present study, we found that miR-128b expression was down-regulated in tissues from 18 GC patients and 3 carcinoma cell lines. In turn, over-expression of miR-128b suppressed GC cell proliferation, invasion and promoted apoptosis. Moreover, miR-128b was predicted to bind the 3'UTR of PDK1 gene using bioinformatic target-screening tools. Accordingly, ectopic expression of miR-128b inhibited the PDK1 expression at both transcriptional and post-transcriptional levels, and furthermore, the expression of gene tailed by the 3'UTR of PDK1 gene was significantly decreased in a dualluciferase reporter assay, suggesting that PDK1 was a direct target of miR-128b in GC cells. In the conditon of miR- 128b over-expression, we also observed spontaneous inactivation of the Akt/NF-κB signalling, implying PDK1 was a potential regulator of this pathway. In conclusion, our study shed some novel light on miR-128b-PDK1/Akt/NF-κB axis on GC progression.

Genome-wide expressions in autologous eutopic and ectopic endometrium of fertile women with endometriosis.

PubMed

Khan, Meraj A; Sengupta, Jayasree; Mittal, Suneeta; Ghosh, Debabrata

2012-09-24

In order to obtain a lead of the pathophysiology of endometriosis, genome-wide expressional analyses of eutopic and ectopic endometrium have earlier been reported, however, the effects of stages of severity and phases of menstrual cycle on expressional profiles have not been examined. The effect of genetic heterogeneity and fertility history on transcriptional activity was also not considered. In the present study, a genome-wide expression analysis of autologous, paired eutopic and ectopic endometrial samples obtained from fertile women (n=18) suffering from moderate (stage 3; n=8) or severe (stage 4; n=10) ovarian endometriosis during proliferative (n=13) and secretory (n=5) phases of menstrual cycle was performed. Individual pure RNA samples were subjected to Agilent's Whole Human Genome 44K microarray experiments. Microarray data were validated (P<0.01) by estimating transcript copy numbers by performing real time RT-PCR of seven (7) arbitrarily selected genes in all samples. The data obtained were subjected to differential expression (DE) and differential co-expression (DC) analyses followed by networks and enrichment analysis, and gene set enrichment analysis (GSEA). The reproducibility of prediction based on GSEA implementation of DC results was assessed by examining the relative expressions of twenty eight (28) selected genes in RNA samples obtained from fresh pool of eutopic and ectopic samples from confirmed ovarian endometriosis patients with stages 3 and 4 (n=4/each) during proliferative and secretory (n=4/each) phases. Higher clustering effect of pairing (cluster distance, cd=0.1) in samples from same individuals on expressional arrays among eutopic and ectopic samples was observed as compared to that of clinical stages of severity (cd=0.5) and phases of menstrual cycle (cd=0.6). Post hoc analysis revealed anomaly in the expressional profiles of several genes associated with immunological, neuracrine and endocrine functions and gynecological cancers however with no overt oncogenic potential in endometriotic tissue. Dys-regulation of three (CLOCK, ESR1, and MYC) major transcription factors appeared to be significant causative factors in the pathogenesis of ovarian endometriosis. A novel cohort of twenty-eight (28) genes representing potential marker for ovarian endometriosis in fertile women was discovered. Dysfunctional expression of immuno-neuro-endocrine behaviour in endometrium appeared critical to endometriosis. Although no overt oncogenic potential was evident, several genes associated with gynecological cancers were observed to be high in the expressional profiles in endometriotic tissue.

Ectopic ERK Expression Induces Phenotypic Conversion of C10 Cells and Alters DNA Methyltransferase Expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sontag, Ryan L.; Weber, Thomas J.

2012-05-04

In some model systems constitutive extracellular signal regulated kinase (ERK) activation is sufficient to promote an oncogenic phenotype. Here we investigate whether constitutive ERK expression influences phenotypic conversion in murine C10 type II alveolar epithelial cells. C10 cells were stably transduced with an ERK1-green fluorescent protein (ERK1-GFP) chimera or empty vector and ectopic ERK expression was associated with the acquisition of soft agar focus-forming potential in late passage, but not early passage cells. Late passage ERK1-GFP cells exhibited a significant increase in the expression of DNA methyl transferases (DNMT1 and 3b) and a marked increase in sensitivity to 5-azacytidine (5-azaC)-mediatedmore » toxicity, relative to early passage ERK1-GFP cells and vector controls. The expression of xeroderma pigmentosum complementation group A (XPA) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were significantly increased in late passage cells, suggesting enhanced DNA damage recognition and repair activity which we interpret as a reflection of genomic instability. Phospho-ERK levels were dramatically decreased in late passage ERK1-GFP cells, relative to early passage and vector controls, and phospho-ERK levels were restored by treatment with sodium orthovanadate, indicating a role for phosphatase activity in this response. Collectively these observations suggest that ectopic ERK expression promotes phenotypic conversion of C10 cells that is associated with latent effects on epigenetic programming and phosphatase activities.« less

Association of nbl gene expression and glucocorticoid-induced apoptosis in mouse thymus in vivo.

PubMed Central

Naora, H; Nishida, T; Shindo, Y; Adachi, M; Naora, H

1995-01-01

A gene of unknown biological function, nbl, was originally isolated by virtue of its abundance in a Namalwa Burkitt Lymphoma cDNA library. nbl expression was initially found to be higher in tissues which exhibited internucleosomal DNA cleavage characteristic of apoptosis, than in tissues which did not exhibit a 'DNA ladder'. nbl expression was therefore examined in mouse thymus in vivo, in which apoptosis is induced by the glucocorticoid, dexamethasone. nbl expression was markedly enhanced by dexamethasone treatment and then sharply decreased prior to the occurrence of maximal 'DNA ladder' formation. In contrast, expression of myc, which is believed to be involved in apoptosis in other cell systems, declined as thymic apoptosis increased. Thymic apoptosis was blocked by the transcriptional inhibitor actinomycin D, if administered when nbl expression was enhanced, but not before or after the peak of nbl expression. These results suggest that nbl expression is associated with thymic apoptosis. Images Figure 1 Figure 3 Figure 4 Figure 6 PMID:7635523

Methylation of PLCD1 and adenovirus-mediated PLCD1 overexpression elicits a gene therapy effect on human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mu, Haixi; Department of Endocrine and breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016; Wang, Na

Our previous study showed that PLCD1 significantly decreases cell proliferation and affects cell cycle progression in breast cancer cells. In the present study, we aimed to investigate its functional and molecular mechanisms, and whether or not can become a new target for gene therapies. We found reduced PLCD1 protein expression in breast tumor tissues compared with paired surgical margin tissues. PLCD1 promoter CpG methylation was detected in 55 of 96 (57%) primary breast tumors, but not in surgical-margin tissues and normal breast tissues. Ectopic expression of PLCD1 inhibited breast tumor cell proliferation in vivo by inducing apoptosis and suppressed tumormore » cell migration by regulating cytoskeletal reorganization proteins including RhoA and phospho-cofilin. Furthermore, we found that PLCD1 induced p53 accumulation, increased p27 and p21 protein levels, and cleaved PARP. Finally, we constructed an adenoviral vector expressing PLCD1 (AdH5-PLCD1), which exhibited strong cytotoxicity in breast cancer cells. Our findings provide insights into the development of PLCD1 gene therapies for breast cancer and perhaps, other human cancers. - Highlights: • PLCD1 is downregulated via hypermethylation in breast cancer. • PLCD1 suppressed cell migration by regulating cytoskeletal reorganization proteins. • Adenovirus AdHu5-PLCD1 may be a novel therapeutic option for breast cancer.« less

The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim

PubMed Central

Jurado, Sabine; Gleeson, Kimberly; O’Donnell, Kristy; Izon, David J.; Walkley, Carl R.; Strasser, Andreas; Tarlinton, David M.

2012-01-01

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis. PMID:22891272

Circular RNA MYLK as a competing endogenous RNA promotes bladder cancer progression through modulating VEGFA/VEGFR2 signaling pathway.

PubMed

Zhong, Zhenyu; Huang, Mengge; Lv, Mengxin; He, Yunfeng; Duan, Changzhu; Zhang, Luyu; Chen, Junxia

2017-09-10

Accumulating evidences indicate that circular RNAs (circRNAs) play a vital role in modulating gene expression. However, the mechanisms underlying circRNAs remain largely elusive. Here, we screened circRNA and mRNA expression profiles of bladder carcinoma (BC) using microarray analysis. We found that circRNA-MYLK and VEGFA were significantly up-regulated and co-expressed in BC. Importantly, circRNA-MYLK levels were related to the progression of stage and grade of BC. Mechanistically, we demonstrated that circRNA-MYLK could directly bind to miR-29a and relieve suppression for target VEGFA, which activated VEGFA/VEGFR2 signaling pathway. Functionally, we found that ectopically expressing circRNA-MYLK accelerated cell proliferation, migration, tube formation of HUVEC and rearranged cytoskeleton. Moreover, up-regulating circRNA-MYLK promoted epithelial-mesenchymal transition (EMT). Whereas circRNA-MYLK knockdown decreased cell proliferation, motility, and induced apoptosis. Finally, up-regulating circRNA-MYLK promoted the growth, angiogenesis and metastasis of BC xenografts. Taken together, this study demonstrated for the first time that circRNA-MYLK might function as competing endogenous RNA (ceRNA) for miR-29a, which could contribute to EMT and the development of BC through activating VEGFA/VEGFR2 and downstream Ras/ERK signaling pathway. Our data suggest that circRNA-MYLK would be a promising target for BC diagnosis and therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway

PubMed Central

Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D.; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn

2017-01-01

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted. PMID:28107519

Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway.

PubMed

Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn; Mai, Chun-Wai

2017-01-01

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted.

Crizotinib induces apoptosis and gene expression changes in ALK+ anaplastic large cell lymphoma cell lines; brentuximab synergizes and doxorubicin antagonizes.

PubMed

Hudson, Sandra; Wang, Dongliang; Middleton, Frank; Nevaldine, Barbara H; Naous, Rana; Hutchison, Robert E

2018-04-26

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) shows 60-70% event free survival with standard treatments. Targeted therapies are being tested for increased benefit and/or reduced toxicity, but interactions with standard agents are not well known. We exposed four ALCL cell lines to two targeted agents, crizotinib and brentuximab vedotin, and to two standard agents, doxorubicin and vinblastine. For each agent and combination, we measured apoptosis and expression of approximately 300 previously annotated genes of interest using targeted RNA-sequencing. An aurora kinase inhibitor, alisertib, was similarly tested for gene expression effects. Only crizotinib, alone or in combination, showed significant effects (adjusted P < 0.05) on expression and apoptosis. One hundred and nine of 277 gene expressions showed crizotinib-associated differential expression, mostly downregulation, 62 associated with apoptosis, and 28 associated with both crizotinib and apoptosis. Doxorubicin was antagonistic with crizotinib on gene expression and apoptosis. Brentuximab was synergistic with crizotinib in apoptosis, and not antagonistic in gene expression. Vinblastine also appeared synergistic with crizotinib but did not achieve statistical significance. Alisertib did not show significant expression changes. Our data suggest that crizotinib induces apoptosis through orderly changes in cell signaling associated with ALK inhibition. Expression effects of crizotinib and associated apoptosis are antagonized by doxorubicin, but apoptosis is synergized by brentuximab vedotin and possibly vinblastine. These findings suggest that concurrent use of crizotinib and doxorubicin may be counterproductive, while the pairing of crizotinib with brentuximab (or vinblastine) may increase efficacy. Alisertib did not induce expression changes at cytotoxic dosage. © 2018 Wiley Periodicals, Inc.

The Circular RNA Interacts with STAT3, Increasing Its Nuclear Translocation and Wound Repair by Modulating Dnmt3a and miR-17 Function.

PubMed

Yang, Zhen-Guo; Awan, Faryal Mehwish; Du, William W; Zeng, Yan; Lyu, Juanjuan; Wu, De; Gupta, Shaan; Yang, Weining; Yang, Burton B

2017-09-06

Delayed or impaired wound healing is a major health issue worldwide, especially in patients with diabetes and atherosclerosis. Here we show that expression of the circular RNA circ-Amotl1 accelerated healing process in a mouse excisional wound model. Further studies showed that ectopic circ-Amotl1 increased protein levels of Stat3 and Dnmt3a. The increased Dnmt3a then methylated the promoter of microRNA miR-17, decreasing miR-17-5p levels but increasing fibronectin expression. We found that Stat3, similar to Dnmt3a and fibronectin, was a target of miR-17-5p. Decreased miR-17-5p levels would increase expression of fibronectin, Dnmt3a, and Stat3. All of these led to increased cell adhesion, migration, proliferation, survival, and wound repair. Furthermore, we found that circ-Amotl1 not only increased Stat3 expression but also facilitated Stat3 nuclear translocation. Thus, the ectopic expressed circ-Amotl1 and Stat3 were mainly translocated to nucleus. In the presence of circ-Amotl1, Stat3 interacted with Dnmt3a promoter with increased affinity, facilitating Dnmt3a transcription. Ectopic application of circ-Amotl1 accelerating wound repair may shed light on skin wound healing clinically. Copyright © 2017. Published by Elsevier Inc.

A Multistate Toggle Switch Defines Fungal Cell Fates and Is Regulated by Synergistic Genetic Cues

PubMed Central

Anderson, Matthew Z.; Porman, Allison M.; Wang, Na; Mancera, Eugenio; Bennett, Richard J.

2016-01-01

Heritable epigenetic changes underlie the ability of cells to differentiate into distinct cell types. Here, we demonstrate that the fungal pathogen Candida tropicalis exhibits multipotency, undergoing stochastic and reversible switching between three cellular states. The three cell states exhibit unique cellular morphologies, growth rates, and global gene expression profiles. Genetic analysis identified six transcription factors that play key roles in regulating cell differentiation. In particular, we show that forced expression of Wor1 or Efg1 transcription factors can be used to manipulate transitions between all three cell states. A model for tristability is proposed in which Wor1 and Efg1 are self-activating but mutually antagonistic transcription factors, thereby forming a symmetrical self-activating toggle switch. We explicitly test this model and show that ectopic expression of WOR1 can induce white-to-hybrid-to-opaque switching, whereas ectopic expression of EFG1 drives switching in the opposite direction, from opaque-to-hybrid-to-white cell states. We also address the stability of induced cell states and demonstrate that stable differentiation events require ectopic gene expression in combination with chromatin-based cues. These studies therefore experimentally test a model of multistate stability and demonstrate that transcriptional circuits act synergistically with chromatin-based changes to drive cell state transitions. We also establish close mechanistic parallels between phenotypic switching in unicellular fungi and cell fate decisions during stem cell reprogramming. PMID:27711197

Spatial distribution of osteoblast-specific transcription factor Cbfa1 and bone formation in atherosclerotic arteries.

PubMed

Bobryshev, Yuri V; Killingsworth, Murray C; Lord, Reginald S A

2008-08-01

The mechanisms of ectopic bone formation in arteries are poorly understood. Osteoblasts might originate either from stem cells that penetrate atherosclerotic plaques from the blood stream or from pluripotent mesenchymal cells that have remained in the arterial wall from embryonic stages of the development. We have examined the frequency of the expression and spatial distribution of osteoblast-specific factor-2/core binding factor-1 (Osf2/Cbfa1) in carotid and coronary arteries. Cbfa1-expressing cells were rarely observed but were found in all tissue specimens in the deep portions of atherosclerotic plaques under the necrotic cores. The deep portions of atherosclerotic plaques under the necrotic cores were characterized by the lack of capillaries of neovascularization. In contrast, plaque shoulders, which were enriched by plexuses of neovascularization, lacked Cbfa1-expressing cells. No bone formation was found in any of the 21 carotid plaques examined and ectopic bone was observed in only two of 12 coronary plaques. We speculate that the sparse invasion of sprouts of neovascularization into areas underlying the necrotic cores, where Cbfa1-expressing cells reside, might explain the rarity of events of ectopic bone formation in the arterial wall. This study has also revealed that Cbfa1-expressing cells contain alpha-smooth muscle actin and myofilaments, indicating their relationship with arterial smooth muscle cells.

The co-expression of GPER and Gankyrin in ovarian endometriosis and its correlation with the rASRM stages.

PubMed

Zhang, Chun; Yuan, Xiying; Zhang, Yi

2016-01-01

The aim of this study was to examine the expression of G protein-coupled estrogen receptor (GPER) and Gankyrin in ovarian endometriosis, analyze their clinicopathological significance, and investigate their correlation. Quantitative real-time polymerase chain reaction and Western blot were performed to testify mRNA and protein expression of GPER and Gankyrin in ovarian endometriosis. Immunohistochemical staining (streptavidin-peroxidase method) was conducted to determine the expression and distribution of GPER and Gankyrin protein in matched ectopic and eutopic endometrium of endometriosis and normal endometrium. We also investigated their associations with rASRM stages and the correlation between the two proteins. GPER and Gankyrin were found overexpressed in ectopic endometrium of endometriosis compared with either its eutopic counterpart or endometrium from normal patients. The immunohistochemical analysis also revealed that higher expression was observed in eutopic endometrium with or without endometriosis during proliferative phase in comparison to secretory phase. These two proteins were positively correlated with the stages of endometriosis. Moreover, a significant positive correlation was found between GPER and Gankyrin both in ectopic and eutopic endometrium of the ovarian endometriosis. GPER and Gankyrin might be implicated in the hormonal regulation of endometriosis and be associated with the severity of endometriosis. In addition, GPER and Gankyrin were found to be positively correlated, which could possibly serve as novel therapeutic targets for this disease.

Vasostatin-2 inhibits cell proliferation and adhesion in vascular smooth muscle cells, which are associated with the progression of atherosclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Jianghong, E-mail: jianghonghou@163.com; Xue, Xiaolin; Li, Junnong

2016-01-22

Recently, the serum expression level of vasostatin-2 was found to be reduced and is being studied as an important indicator to assess the presence and severity of coronary artery disease; the functional properties of vasostatin-2 and its relationship with the development of atherosclerosis remains unclear. In this study, we attempted to detect the expression of vasostatin-2 and its impact on human vascular smooth muscle cells (VSMCs). Quantitative real-time PCR (qRT-PCR) and western blot were used to assess the expression level of vasostatin-2 in VSMCs between those from atherosclerosis and disease-free donors; we found that vasostatin-2 was significantly down-regulated in atherosclerosismore » patient tissues and cell lines. In addition, the over-expression of vasostatin-2 apparently inhibits cell proliferation and migration in VSMCs. Gain-of-function in vitro experiments further show that vasostatin-2 over-expression significantly inhibits inflammatory cytokines release in VSMCs. In addition, cell adhesion experimental analysis showed that soluble adhesion molecules (sICAM-1, sVCAM-1) had decreased expression when vasostatin-2 was over-expressed in VSMCs. Therefore, our results indicate that vasostatin-2 is an atherosclerosis-related factor that can inhibit cell proliferation, inflammatory response and cell adhesion in VSMCs. Taken together, our results indicate that vasostatin-2 could serve as a potential diagnostic biomarker and therapeutic option for human atherosclerosis in the near future. - Highlights: • Vasostatin-2 levels were down-regulated in atherosclerosis patient tissues and VSMCs. • Ectopic expression of vasostatin-2 directly affects cell proliferation and migration in vitro. • Ectopic expression of vasostatin-2 protein affects pro-inflammatory cytokines release in VSMCs. • Ectopic expression of vasostatin-2 protein affects cell adhesion in VSMCs.« less

Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L).

PubMed

Srinivasan, C; Liu, Zongrang; Scorza, Ralph

2011-04-01

Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

Ectopic High Expression of E2-EPF Ubiquitin Carrier Protein Indicates a More Unfavorable Prognosis in Brain Glioma.

PubMed

Zhang, Xiaohui; Zhao, Fangbo; Zhang, Shujun; Song, Yichun

2017-04-01

Ubiquitination of proteins meant for elimination is a primary method of eukaryotic cellular protein degradation. The ubiquitin carrier protein E2-EPF is a key degradation enzyme that is highly expressed in many tumors. However, its expression and prognostic significance in brain glioma are still unclear. The aim of this study was to reveal how the level of E2-EPF relates to prognosis in brain glioma. Thirty low-grade and 30 high-grade brain glioma samples were divided into two tissue microarrays each. Levels of E2-EPF protein were examined by immunohistochemistry and immunofluorescence. Quantitative real-time polymerase chain reaction was used to analyze the level of E2-EPF in 60 glioma and 3 normal brain tissue samples. The relationship between E2-EPF levels and prognosis was analyzed by Kaplan-Meier survival curves. E2-EPF levels were low in normal brain tissue samples but high in glioma nuclei. E2-EPF levels gradually increased as glioma grade increased (p < 0.05). Ectopic E2-EPF levels in high-grade glioma were significantly higher than in low-grade glioma (p < 0.01). The 5-year survival rate of glioma patients with high E2-EPF levels was shorter than in patients with low expression (p < 0.05). Furthermore, the 5-year survival rate of patients with ectopic E2-EPF was significantly shorter than patients with only nuclear E2-EPF (p < 0.01). These results suggest that higher E2-EPF levels, especially ectopic, are associated with higher grade glioma and shorter survival. E2-EPF levels may play a key role in predicting the prognosis for patients with brain glioma.

Alteration of flower color in Iris germanica L. 'Fire Bride' through ectopic expression of phytoene synthase gene (crtB) from Pantoea agglomerans.

PubMed

Jeknić, Zoran; Jeknić, Stevan; Jevremović, Slađana; Subotić, Angelina; Chen, Tony H H

2014-08-01

Genetic modulation of the carotenogenesis in I. germanica 'Fire Bride' by ectopic expression of a crtB gene causes several flower parts to develop novel orange and pink colors. Flower color in tall bearded irises (Iris germanica L.) is determined by two distinct biochemical pathways; the carotenoid pathway, which imparts yellow, orange and pink hues and the anthocyanin pathway, which produces blue, violet and maroon flowers. Red-flowered I. germanica do not exist in nature and conventional breeding methods have thus far failed to produce them. With a goal of developing iris cultivars with red flowers, we transformed a pink iris I. germanica, 'Fire Bride', with a bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under the control of the promoter region of a gene for capsanthin-capsorubin synthase from Lilium lancifolium (Llccs). This approach aimed to increase the flux of metabolites into the carotenoid biosynthetic pathway and lead to elevated levels of lycopene and darker pink or red flowers. Iris callus tissue ectopically expressing the crtB gene exhibited a color change from yellow to pink-orange and red, due to accumulation of lycopene. Transgenic iris plants, regenerated from the crtB-transgenic calli, showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink), while the standards and falls showed no significant differences in color when compared to control plants. HPLC and UHPLC analysis confirmed that the color changes were primarily due to the accumulation of lycopene. In this study, we showed that ectopic expression of a crtB can be used to successfully alter the color of certain flower parts in I. germanica 'Fire Bride' and produce new flower traits.

Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo.

PubMed

De Bari, Cosimo; Dell'Accio, Francesco; Luyten, Frank P

2004-01-01

We previously reported the identification in a nude mouse assay of molecular markers predictive of the capacity of articular cartilage-derived cells (ACDCs) to form ectopic stable cartilage that is resistant to vascular invasion and endochondral ossification. In the present study, we investigated whether in vitro-differentiated mesenchymal stem cells (MSCs) from the synovial membrane (SM) express the stable-chondrocyte markers and form ectopic stable cartilage in vivo. Chondrogenesis was induced in micromass culture with the addition of transforming growth factor beta1 (TGFbeta1). After acquisition of the cartilage phenotype, micromasses were implanted subcutaneously into nude mice. Alternatively, cells were released enzymatically and either replated in monolayer or injected intramuscularly into nude mice. Marker analysis was performed by quantitative reverse transcription-polymerase chain reaction. Cell death was detected with TUNEL assay. Cartilage-like micromasses and released cells expressed the stable-chondrocyte markers at levels comparable with those expressed by stable ACDCs. The released cells lost chondrocyte marker expression by 24 hours in monolayer and failed to form cartilage when injected intramuscularly into nude mice. Instead, myogenic differentiation was detected. When intact TGFbeta1-treated micromasses were implanted subcutaneously, they partially lost their cartilage phenotype and underwent cell death and neoangiogenesis within 1 week. At later time points (15-40 days), we retrieved neither cartilage nor bone, and human cells were not detectable. The chondrocyte-like phenotype of human SM MSCs, induced in vitro under specific conditions, appears to be unstable and is not sufficient to obtain ectopic formation of stable cartilage in vivo. Studies in animal models of joint surface defect repair are necessary to evaluate the stability of the SM MSC chondrocyte-like phenotype within the joint environment.

Transient development of ovotestes in XX Sox9 transgenic mice

PubMed Central

Gregoire, Elodie P.; Lavery, Rowena; Chassot, Anne-Amandine; Akiyama, Haruhiko; Treier, Mathias; Behringer, Richard R.; Chaboissier, Marie-Christine

2010-01-01

The sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice, induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9Tg/+ gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads, and is able to induce the expression of EGFP, knocked into the 3′ UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9-EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX foetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth. PMID:20965161

Loss of Dickkopf 3 Promotes the Tumorigenesis of Basal Breast Cancer

PubMed Central

Lorsy, Eva; Topuz, Aylin Sophie; Geisler, Cordelia; Stahl, Sarah; Garczyk, Stefan; von Stillfried, Saskia; Hoss, Mareike; Gluz, Oleg; Hartmann, Arndt; Knüchel, Ruth; Dahl, Edgar

2016-01-01

Dickkopf 3 (DKK3) has been associated with tumor suppression of various tumor entities including breast cancer. However, the functional impact of DKK3 on the tumorigenesis of distinct molecular breast cancer subtypes has not been considered so far. Therefore, we initiated a study analyzing the subtype-specific DKK3 expression pattern as well as its prognostic and functional impact with respect to breast cancer subtypes. Based on three independent tissue cohorts including one in silico dataset (n = 30, n = 463 and n = 791) we observed a clear down-regulation of DKK3 expression in breast cancer samples compared to healthy breast tissue controls on mRNA and protein level. Interestingly, most abundant reduction of DKK3 expression was detected in the highly aggressive basal breast cancer subtype. Analyzing a large in silico dataset comprising 3,554 cases showed that low DKK3 mRNA expression was significantly associated with reduced recurrence free survival (RFS) of luminal and basal-like breast cancer cases. Functionally, DKK3 re-expression in human breast cancer cell lines led to suppression of cell growth possibly mediated by up-regulation of apoptosis in basal-like but not in luminal-like breast cancer cell lines. Moreover, ectopic DKK3 expression in mesenchymal basal breast cancer cells resulted in partial restoration of epithelial cell morphology which was molecularly supported by higher expression of epithelial markers like E-Cadherin and down-regulation of mesenchymal markers such as Snail 1. Hence, we provide evidence that down-regulation of DKK3 especially promotes tumorigenesis of the aggressive basal breast cancer subtype. Further studies decoding the underlying molecular mechanisms of DKK3-mediated effects may help to identify novel targeted therapies for this clinically highly relevant breast cancer subtype. PMID:27467270

Ectopic expression of Arabidopsis broad-spectrum resistance gene RPW8.2 improves the resistance to powdery mildew in grapevine (Vitis vinifera).

PubMed

Hu, Yang; Li, Yajuan; Hou, Fengjuan; Wan, Dongyan; Cheng, Yuan; Han, Yongtao; Gao, Yurong; Liu, Jie; Guo, Ye; Xiao, Shunyuan; Wang, Yuejin; Wen, Ying-Qiang

2018-02-01

Powdery mildew is the most economically important disease of cultivated grapevines worldwide. Here, we report that the Arabidopsis broad-spectrum disease resistance gene RPW8.2 could improve resistance to powdery mildew in Vitis vinifera cv. Thompson Seedless. The RPW8.2-YFP fusion gene was stably expressed in grapevines from either the constitutive 35S promoter or the native promoter (NP) of RPW8.2. The grapevine shoots and plantlets transgenic for 35S::RPW8.2-YFP showed reduced rooting and reduced growth at later development stages in the absence of any pathogens. Infection tests with an adapted grapevine powdery mildew isolate En NAFU1 showed that hyphal growth and sporulation were significantly restricted in transgenic grapevines expressing either of the two constructs. The resistance appeared to be attributable to the ectopic expression of RPW8.2, and associated with the enhanced encasement of the haustorial complex (EHC) and onsite accumulation of H 2 O 2 . In addition, the RPW8.2-YFP fusion protein showed focal accumulation around the fungal penetration sites. Transcriptome analysis revealed that ectopic expression of RPW8.2 in grapevines not only significantly enhanced salicylic acid-dependent defense signaling, but also altered expression of other phytohormone-associated genes. Taken together, our results indicate that RPW8.2 could be utilized as a transgene for improving resistance against powdery mildew in grapevines. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Jing-Ping; Lin, Kai-Han; Liu, Chun-Yen

In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutantmore » p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. - Highlights: • Teroxirone repressed tumor cell growth in nude mice of human lung cancer cells. • The apoptotic cell death reverted by caspase-3 inhibitor is related to p53 status. • Teroxirone provides a good candidate for lung cancer treatment.« less

Soluble VEGF isoforms are essential for establishingepiphyseal vascularization and regulating chondrocyte development and survival

PubMed Central

Maes, Christa; Stockmans, Ingrid; Moermans, Karen; Van Looveren, Riet; Smets, Nico; Carmeliet, Peter; Bouillon, Roger; Carmeliet, Geert

2004-01-01

VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF120, VEGF164, and VEGF188 isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF164 or only VEGF188 (in VEGF188/188 mice) was sufficient for metaphyseal development. VEGF188/188 mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF188 isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF188/188 mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF188 isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation. PMID:14722611

Enhanced regeneration potential of mobilized dental pulp stem cells from immature teeth.

PubMed

Nakayama, H; Iohara, K; Hayashi, Y; Okuwa, Y; Kurita, K; Nakashima, M

2017-07-01

We have previously demonstrated that dental pulp stem cells (DPSCs) isolated from mature teeth by granulocyte colony-stimulating factor (G-CSF)-induced mobilization method can enhance angiogenesis/vasculogenesis and improve pulp regeneration when compared with colony-derived DPSCs. However, the efficacy of this method in immature teeth with root-formative stage has never been investigated. Therefore, the aim of this study was to examine the stemness, biological characteristics, and regeneration potential in mobilized DPSCs compared with colony-derived DPSCs from immature teeth. Mobilized DPSCs isolated from immature teeth were compared to colony-derived DPSCs using methods including flow cytometry, migration assays, mRNA expression of angiogenic/neurotrophic factor, and induced differentiation assays. They were also compared in trophic effects of the secretome. Regeneration potential was further compared in an ectopic tooth transplantation model. Mobilized DPSCs had higher migration ability and expressed more angiogenic/neurotrophic factors than DPSCs. The mobilized DPSC secretome produced a higher stimulatory effect on migration, immunomodulation, anti-apoptosis, endothelial differentiation, and neurite extension. In addition, vascularization and pulp regeneration potential were higher in mobilized DPSCs than in DPSCs. G-CSF-induced mobilization method enhances regeneration potential of colony-derived DPSCs from immature teeth. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Soluble VEGF isoforms are essential for establishing epiphyseal vascularization and regulating chondrocyte development and survival.

PubMed

Maes, Christa; Stockmans, Ingrid; Moermans, Karen; Van Looveren, Riet; Smets, Nico; Carmeliet, Peter; Bouillon, Roger; Carmeliet, Geert

2004-01-01

VEGF is crucial for metaphyseal bone vascularization. In contrast, the angiogenic factors required for vascularization of epiphyseal cartilage are unknown, although this represents a developmentally and clinically important aspect of bone growth. The VEGF gene is alternatively transcribed into VEGF(120), VEGF(164), and VEGF(188) isoforms that differ in matrix association and receptor binding. Their role in bone development was studied in mice expressing single isoforms. Here we report that expression of only VEGF(164) or only VEGF(188) (in VEGF(188/188) mice) was sufficient for metaphyseal development. VEGF(188/188) mice, however, showed dwarfism, disrupted development of growth plates and secondary ossification centers, and knee joint dysplasia. This phenotype was at least partly due to impaired vascularization surrounding the epiphysis, resulting in ectopically increased hypoxia and massive chondrocyte apoptosis in the interior of the epiphyseal cartilage. In addition to the vascular defect, we provide in vitro evidence that the VEGF(188) isoform alone is also insufficient to regulate chondrocyte proliferation and survival responses to hypoxia. Consistent herewith, chondrocytes in or close to the hypoxic zone in VEGF(188/188) mice showed increased proliferation and decreased differentiation. These findings indicate that the insoluble VEGF(188) isoform is insufficient for establishing epiphyseal vascularization and regulating cartilage development during endochondral bone formation.

GADD45α sensitizes cervical cancer cells to radiotherapy via increasing cytoplasmic APE1 level.

PubMed

Li, Qing; Wei, Xi; Zhou, Zhi-Wei; Wang, Shu-Nan; Jin, Hua; Chen, Kui-Jun; Luo, Jia; Westover, Kenneth D; Wang, Jian-Min; Wang, Dong; Xu, Cheng-Xiong; Shan, Jin-Lu

2018-05-09

Radioresistance remains a major clinical challenge in cervical cancer therapy. However, the mechanism for the development of radioresistance in cervical cancer is unclear. Herein, we determined that growth arrest and DNA-damage-inducible protein 45α (GADD45α) is decreased in radioresistant cervical cancer compared to radiosensitive cancer both in vitro and in vivo. In addition, silencing GADD45α prevents cervical cancer cells from undergoing radiation-induced DNA damage, cell cycle arrest, and apoptosis. More importantly, our data show that the overexpression of GADD45α significantly enhances the radiosensitivity of radioresistant cervical cancer cells. These data show that GADD45α decreases the cytoplasmic distribution of APE1, thereby enhancing the radiosensitivity of cervical cancer cells. Furthermore, we show that GADD45α inhibits the production of nitric oxide (NO), a nuclear APE1 export stimulator, by suppressing both endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) in cervical cancer cells. In conclusion, our findings suggest that decreased GADD45α expression significantly contributes to the development of radioresistance and that ectopic expression of GADD45α sensitizes cervical cancer cells to radiotherapy. GADD45α inhibits the NO-regulated cytoplasmic localization of APE1 through inhibiting eNOS and iNOS, thereby enhancing the radiosensitivity of cervical cancer cells.

A type III effector antagonizes death receptor signalling during bacterial gut infection.

PubMed

Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Lung, Tania Wong Fok; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare V L; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O'Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

2013-09-12

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.

Trypanosoma cruzi trans-sialidase: A potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling

PubMed Central

Chuenkova, Marina V.; Furnari, Frank B.; Cavenee, Webster K.; Pereira, Miercio A.

2001-01-01

Patients infected with Trypanosoma cruzi may remain asymptomatic for decades and show signs of neuroregeneration in the peripheral nervous system (PNS). In the absence of such neuroregeneration, patients may die in part by extensive neuronal destruction in the gastrointestinal tract. Thus, T. cruzi may, despite their invasion of the PNS, directly prevent cell death to keep nerve destruction in check. Indeed, T. cruzi invasion of Schwann cells, their prime target in PNS, suppressed host-cell apoptosis caused by growth-factor deprivation. The trans-sialidase (TS) of T. cruzi and the Cys-rich domain of TS reproduced the antiapoptotic activity of the parasites at doses (≥3.0 nM) comparable or lower than those of bona fide mammalian growth factors. This effect was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). TS also activated Akt, a downstream effector of PI3K. Ectopic expression of TS in an unrelated parasite, Leishmania major, turned those parasites into activators of Akt in Schwann cells. In contrast, the Cys-rich domain of TS did not block apoptosis in Schwann cells overexpressing dominant-negative Akt or constitutively active PTEN, a negative regulator of PI3K/Akt signaling. The results demonstrate that T. cruzi, through its TS, triggers the survival of host Schwann cells via the PI3K/Akt pathway, suggesting a role for PI3K/Akt in the pathogenesis of Chagas' disease. PMID:11481434

The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

PubMed

Jordan, K C; Clegg, N J; Blasi, J A; Morimoto, A M; Sen, J; Stein, D; McNeill, H; Deng, W M; Tworoger, M; Ruohola-Baker, H

2000-04-01

Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

Tendon Mineralization Is Progressive and Associated with Deterioration of Tendon Biomechanical Properties, and Requires BMP-Smad Signaling in the Mouse Achilles Tendon Injury Model

PubMed Central

Zhang, Kairui; Asai, Shuji; Hast, Michael W.; Liu, Min; Usami, Yu; Iwamoto, Masahiro; Soslowsky, Louis J.; Enomoto-Iwamoto, Motomi

2016-01-01

Ectopic tendon mineralization can develop following tendon rupture or trauma surgery. The pathogenesis of ectopic tendon mineralization and its clinical impact have not been fully elucidated yet. In this study, we utilized a mouse Achilles tendon injury model to determine whether ectopic tendon mineralization alters the biomechanical properties of the tendon and whether BMP signaling is involved in this condition. A complete transverse incision was made at the midpoint of the right Achilles tendon in 8-week-old CD1 mice and the gap was left open. Ectopic cartilaginous mass formation was found in the injured tendon by 4 weeks post-surgery and ectopic mineralization was detected at 8–10 weeks post-surgery. Ectopic mineralization grew over time and volume of the mineralized materials of 25-weeks samples was about 2.5 fold bigger than that of 10-weeks samples, indicating that injury-induced ectopic tendon mineralization is progressive. In vitro mechanical testing showed that max force, max stress and mid-substance modulus in the 25-weeks samples were significantly lower than the 10-weeks samples. We observed substantial increases in expression of bone morphogenetic protein family genes in injured tendons 1 week post-surgery. Immunohistochemical analysis showed that phosphorylation of both Smad1 and Smad3 were highly increased in injured tendons as early as 1 week post-injury and remained high in ectopic chondrogenic lesions 4 weeks post-injury. Treatment with the BMP receptor kinase inhibitor (LDN193189) significantly inhibited injury-induced tendon mineralization. These findings indicate that injury-induced ectopic tendon mineralization is progressive, involves BMP signaling and associated with deterioration of tendon biomechanical properties. PMID:26825318

Distal-less regulates eyespot patterns and melanization in Bicyclus butterflies.

PubMed

Monteiro, Antónia; Chen, Bin; Ramos, Diane M; Oliver, Jeffrey C; Tong, Xiaoling; Guo, Min; Wang, Wen-Kai; Fazzino, Lisa; Kamal, Firdous

2013-07-01

Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether over-expression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies. Copyright © 2013 Wiley Periodicals, Inc.

Identification of apoptosis-related PLZF target genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernardo, Maria Victoria; Yelo, Estefania; Gimeno, Lourdes

2007-07-27

The PLZF gene encodes a BTB/POZ-zinc finger-type transcription factor, involved in physiological development, proliferation, differentiation, and apoptosis. In this paper, we investigate proliferation, survival, and gene expression regulation in stable clones from the human haematopoietic K562, DG75, and Jurkat cell lines with inducible expression of PLZF. In Jurkat cells, but not in K562 and DG75 cells, PLZF induced growth suppression and apoptosis in a cell density-dependent manner. Deletion of the BTB/POZ domain of PLZF abrogated growth suppression and apoptosis. PLZF was expressed with a nuclear speckled pattern distinctively in the full-length PLZF-expressing Jurkat clones, suggesting that the nuclear speckled localizationmore » is required for PLZF-induced apoptosis. By microarray analysis, we identified that the apoptosis-inducer TP53INP1, ID1, and ID3 genes were upregulated, and the apoptosis-inhibitor TERT gene was downregulated. The identification of apoptosis-related PLZF target genes may have biological and clinical relevance in cancer typified by altered PLZF expression.« less

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

PubMed

Wang, Xiuli; Cui, Fuai; Madhu, Vedavathi; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

2011-02-01

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

Planar cell polarity controls directional Notch signaling in the Drosophila leg

PubMed Central

Capilla, Amalia; Johnson, Ruth; Daniels, Maki; Benavente, María; Bray, Sarah J.; Galindo, Máximo Ibo

2012-01-01

The generation of functional structures during development requires tight spatial regulation of signaling pathways. Thus, in Drosophila legs, in which Notch pathway activity is required to specify joints, only cells distal to ligand-producing cells are capable of responding. Here, we show that the asymmetric distribution of planar cell polarity (PCP) proteins correlates with this spatial restriction of Notch activation. Frizzled and Dishevelled are enriched at distal sides of each cell and hence localize at the interface with ligand-expressing cells in the non-responding cells. Elimination of PCP gene function in cells proximal to ligand-expressing cells is sufficient to alleviate the repression, resulting in ectopic Notch activity and ectopic joint formation. Mutations that compromise a direct interaction between Dishevelled and Notch reduce the efficacy of repression. Likewise, increased Rab5 levels or dominant-negative Deltex can suppress the ectopic joints. Together, these results suggest that PCP coordinates the spatial activity of the Notch pathway by regulating endocytic trafficking of the receptor. PMID:22736244

High glucose induces apoptosis via upregulation of Bim expression in proximal tubule epithelial cells.

PubMed

Zhang, Xiao-Qian; Dong, Jian-Jun; Cai, Tian; Shen, Xue; Zhou, Xiao-Jun; Liao, Lin

2017-04-11

Diabetic nephropathy is the primary cause of end-stage renal disease. Apoptosis of tubule epithelial cells is a major feature of diabetic nephropathy. The mechanisms of high glucose (HG) induced apoptosis are not fully understood. Here we demonstrated that, HG induced apoptosis via upregulating the expression of proapoptotic Bcl-2 homology domain 3 (BH3)-only protein Bim protein, but not bring a significant change in the baseline level of autophagy in HK2 cells. The increase of Bim expression was caused by the ugregulation of transcription factors, FOXO1 and FOXO3a. Bim expression initiates BAX/BAK-mediated mitochondria-dependent apoptosis. Silence of Bim by siRNA in HK2 cells prevented HG-induced apoptosis and also sensitized HK2 cells to autophagy during HG treatment. The autophagy inhibitor 3-MA increased the injury in Bim knockdown HK2 cells by retriggering apoptosis. The above results suggest a Bim-independent apoptosis pathway in HK2 cells, which normally could be inhibited by autophagy. Overall, our results indicate that HG induces apoptosis via up-regulation of Bim expression in proximal tubule epithelial cells.

Specification of spatial identities of cerebellar neuron progenitors by ptf1a and atoh1 for proper production of GABAergic and glutamatergic neurons.

PubMed

Yamada, Mayumi; Seto, Yusuke; Taya, Shinichiro; Owa, Tomoo; Inoue, Yukiko U; Inoue, Takayoshi; Kawaguchi, Yoshiya; Nabeshima, Yo-Ichi; Hoshino, Mikio

2014-04-02

In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.

Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging

PubMed Central

2012-01-01

Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. Results We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. Conclusion We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species. PMID:22780875

Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness

PubMed Central

Siemens, Helge; Jackstadt, Rene; Kaller, Markus; Hermeking, Heiko

2013-01-01

The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches. PMID:24009080

Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis

PubMed Central

Audebert, Stéphane; Helmbacher, Françoise; Dono, Rosanna; Maina, Flavio

2015-01-01

The successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF) receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based Rosa26 stopMet knock-in context (Del-R26 Met) reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesenchyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an RTK when occurring in its endogenous domain of activity. PMID:26393505

Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging.

PubMed

Kumar, Gautam; Kushwaha, Hemant Ritturaj; Panjabi-Sabharwal, Vaishali; Kumari, Sumita; Joshi, Rohit; Karan, Ratna; Mittal, Shweta; Pareek, Sneh L Singla; Pareek, Ashwani

2012-07-10

Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species.

HAT1 induces lung cancer cell apoptosis via up regulating Fas.

PubMed

Han, Na; Shi, Lei; Guo, Qiuyun; Sun, Wei; Yu, Yang; Yang, Li; Zhang, Xiaoxi; Zhang, Mengxian

2017-10-27

The dysfunction of apoptosis is one of the factors contributing to lung cancer (LC) growth. Histone acetyltransferase HAT1 can up regulate cell apoptosis. This study aims to investigate the mechanism by which HAT1 induces LC cell (LCC) apoptosis via up regulating the expression of Fas. In this study, the surgically removed human LC tissues were collected. LCCs were isolated from the LC tissues and analyzed for the expression of HAT1 and Fas by RT-qPCR and Western blotting. We observed that the expression of Fas was negatively correlated with PAR2 in LCCs. Activation of PAR2 suppressed the expression of Fas in normal lung epithelial cells. The expression of HAT1 was lower and positively correlated with Fas expression and negatively correlated with PAR2 expression in LCCs. Activation of PAR2 suppressed Fas expression in lung epithelial cells via inhibiting HAT1. Restoration of HAT1 expression restored Fas expression in LCCs and induced LCC apoptosis. In conclusion, less expression of HAT1 in LCCs was associated with the pathogenesis of LC. Up regulation of HAT1 expression in LCCs can induce LCCs apoptosis, which may be a potential novel therapy for the treatment of LC.

Down-regulated RPS3a/nbl expression during retinoid-induced differentiation of HL-60 cells: a close association with diminished susceptibility to actinomycin D-stimulated apoptosis.

PubMed

Russell, L; Naora, H; Naora, H

2000-04-01

The efficacy of anticancer agents significantly depends on the differential susceptibility of undifferentiated cancer cells and differentiated normal cells to undergo apoptosis. We previously found that enhanced expression of RPS3a/nbl, which apparently encodes a ribosomal protein, seems to prime cells for apoptosis, while suppressing such enhanced expression triggers cell death. The present study found that HL-60 cells induced to differentiate by all-trans retinoic acid did not undergo apoptosis following treatment with actinomycin D whereas undifferentiated HL-60 cells were highly apoptosis-susceptible, confirming earlier suggestions that differentiated cells have diminished apoptosis-susceptibility. Undifferentiated HL-60 cells highly expressed RPS3a/nbl whereas all-trans retinoic acid -induced differentiated cells exhibited markedly reduced levels, suggesting that apoptosis-resistance of differentiated cells could be due to low RPS3a/nbl expression. Down-regulation of enhanced RPS3a/nbl expression was also observed in cells induced to differentiate with the retinoid 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1- propenyl]benzoic acid without any significant induction of cell death. While down-regulation of RPS3a/nbl expression during differentiation did not apparently induce apoptosis, RPS3a/nbl antisense oligomers triggered death of undifferentiated HL-60 cells, but not of retinoid-induced differentiated cells. It therefore seems that while down-regulation of enhanced RPS3a/nbl expression can induce apoptosis in undifferentiated cells, down-regulation of enhanced RPS3a/nbl expression during differentiation occurs independently of apoptosis, and could be regarded as reverting the primed condition to the unprimed (low RPS3a/nbl) state.

E3 ubiquitin ligase Mule ubiquitinates Miz1 and is required for TNFalpha-induced JNK activation.

PubMed

Yang, Yi; Do, HanhChi; Tian, Xuejun; Zhang, Chaozheng; Liu, Xinyuan; Dada, Laura A; Sznajder, Jacob I; Liu, Jing

2010-07-27

The zinc finger transcription factor Miz1 is a negative regulator of TNFalpha-induced JNK activation and cell death through inhibition of TRAF2 K63-polyubiquitination in a transcription-independent manner. Upon TNFalpha stimulation, Miz1 undergoes K48-linked polyubiquitination and proteasomal degradation, thereby relieving its inhibition. However, the underling regulatory mechanism is not known. Here, we report that HECT-domain-containing Mule is the E3 ligase that catalyzes TNFalpha-induced Miz1 polyubiquitination. Mule is a Miz1-associated protein and catalyzes its K48-linked polyubiquitination. TNFalpha-induced polyubiquitination and degradation of Miz1 were inhibited by silencing of Mule and were promoted by ectopic expression of Mule. The interaction between Mule and Miz1 was promoted by TNFalpha independently of the pox virus and zinc finger domain of Miz1. Silencing of Mule stabilized Miz1, thereby suppressing TNFalpha-induced JNK activation and cell death. Thus, our study reveals a molecular mechanism by which Mule regulates TNFalpha-induced JNK activation and apoptosis by catalyzing the polyubiquitination of Miz1.

Retinoic acid temporally orchestrates colonization of the gut by vagal neural crest cells.

PubMed

Uribe, Rosa A; Hong, Stephanie S; Bronner, Marianne E

2018-01-01

The enteric nervous system arises from neural crest cells that migrate as chains into and along the primitive gut, subsequently differentiating into enteric neurons and glia. Little is known about the mechanisms governing neural crest migration en route to and along the gut in vivo. Here, we report that Retinoic Acid (RA) temporally controls zebrafish enteric neural crest cell chain migration. In vivo imaging reveals that RA loss severely compromises the integrity and migration of the chain of neural crest cells during the window of time window when they are moving along the foregut. After loss of RA, enteric progenitors accumulate in the foregut and differentiate into enteric neurons, but subsequently undergo apoptosis resulting in a striking neuronal deficit. Moreover, ectopic expression of the transcription factor meis3 and/or the receptor ret, partially rescues enteric neuron colonization after RA attenuation. Collectively, our findings suggest that retinoic acid plays a critical temporal role in promoting enteric neural crest chain migration and neuronal survival upstream of Meis3 and RET in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Intra-tumoral delivery of functional ID4 protein via PCL/maltodextrin nano-particle inhibits prostate cancer growth

PubMed Central

Morton, Derrick; Sharma, Pankaj; Gorantla, Yamini; Joshi, Jugal; Nagappan, Perri; Pallaniappan, Ravi; Chaudhary, Jaideep

2016-01-01

ID4, a helix loop helix transcriptional regulator has emerged as a tumor suppressor in prostate cancer. Epigenetic silencing of ID4 promotes prostate cancer whereas ectopic expression in prostate cancer cell lines blocks cancer phenotype. To directly investigate the anti-tumor property, full length human recombinant ID4 encapsulated in biodegradable Polycaprolactone/Maltodextrin (PCL-MD) nano-carrier was delivered to LNCaP cells in which the native ID4 was stably silenced (LNCaP(-)ID4). The cellular uptake of ID4 resulted in increased apoptosis, decreased proliferation and colony formation. Intratumoral delivery of PCL-MD ID4 into growing LNCaP(-)ID4 tumors in SCID mice significantly reduced the tumor volume compared to the tumors treated with chemotherapeutic Docetaxel. The study supports the feasibility of using nano-carrier encapsulated ID4 protein as a therapeutic. Mechanistically, ID4 may assimilate multiple regulatory pathways for example epigenetic re-programming, integration of multiple AR co-regulators or signaling pathways resulting in tumor suppressor activity of ID4. PMID:27487149

Mitochondrial peroxiredoxins are essential in regulating the relationship between Drosophila immunity and aging

PubMed Central

Odnokoz, Olena; Nakatsuka, Kyle; Klichko, Vladimir I.; Nguyen, Jacqueline; Solis, Liz Calderon; Ostling, Kaitlin; Badinloo, Marziyeh; Orr, William C.; Radyuk, Svetlana N.

2016-01-01

Previously, we have shown that flies under-expressing the two mitochondrial peroxiredoxins (Prxs), dPrx3 and dPrx5, display increases in tissue-specific apoptosis and dramatically shortened life span, associated with a redox crisis, manifested as changes in GSH:GSSG and accumulation of protein mixed disulfides. To identify specific pathways responsible for the observed biological effects, we performed a transcriptome analysis. Functional clustering revealed a prominent group enriched for immunity-related genes, including a considerable number of NF-kB-dependent antimicrobial peptides (AMP) that are up-regulated in the Prx double mutant. Using qRT-PCR analysis we determined that the age-dependent changes in AMP levels in mutant flies were similar to those observed in controls when scaled to percentage of life span. To further clarify the role of Prx-dependent mitochondrial signaling, we expressed different forms of dPrx5, which unlike the uniquely mitochondrial dPrx3 is found in multiple subcellular compartments, including mitochondrion, nucleus and cytosol. Ectopic expression of dPrx5 in mitochondria but not nucleus or cytosol partially extended longevity under normal or oxidative stress conditions while complete restoration of life span occurred when all three forms of dPrx5 were expressed from the wild type dPrx5 transgene. When dPrx5 was expressed in mitochondria or in all three compartments, it substantially delayed the development of hyperactive immunity while expression of cytosolic or nuclear forms had no effect on the immune phenotype. The data suggest a critical role of mitochondria in development of chronic activation of the immune response triggered by impaired redox control. PMID:27770625

Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

PubMed

Schweitzer, Brock L; Huang, Kelly J; Kamath, Meghana B; Emelyanov, Alexander V; Birshtein, Barbara K; DeKoter, Rodney P

2006-08-15

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

Expression and Functional Pathway Analysis of Nuclear Receptor NR2F2 in Ovarian Cancer

PubMed Central

Hawkins, Shannon M.; Loomans, Holli A.; Wan, Ying-Wooi; Ghosh-Choudhury, Triparna; Coffey, Donna; Xiao, Weimin; Liu, Zhandong; Sangi-Haghpeykar, Haleh

2013-01-01

Context: Recent evidence implicates the orphan nuclear receptor, nuclear receptor subfamily 2, group F, member 2 (NR2F2; chicken ovalbumin upstream promoter-transcription factor II) as both a master regulator of angiogenesis and an oncogene in prostate and other human cancers. Objective: The objective of the study was to determine whether NR2F2 plays a role in ovarian cancer and dissect its potential mechanisms of action. Design, Setting, and Patients: We examined NR2F2 expression in healthy ovary and ovarian cancers using quantitative PCR and immunohistochemistry. NR2F2 expression was targeted in established ovarian cancer cell lines to assess the impact of dysregulated NR2F2 expression in the epithelial compartment of ovarian cancers. Results: Our results indicate that NR2F2 is robustly expressed in the stroma of healthy ovary with little or no expression in epithelia lining the ovarian surface, clefts, or crypts. This pattern of NR2F2 expression was markedly disrupted in ovarian cancers, in which decreased levels of stromal expression and ectopic epithelial expression were frequently observed. Ovarian cancers with the most disrupted patterns of NR2F2 were associated with significantly shorter disease-free interval by Kaplan-Meier analysis. Targeting NR2F2 expression in established ovarian cancer cell lines enhanced apoptosis and increased proliferation. In addition, we found that NR2F2 regulates the expression of NEK2, RAI14, and multiple other genes involved in the cell cycle, suggesting potential pathways by which dysregulated expression of NR2F2 impacts ovarian cancer. Conclusions: These results uncover novel roles for NR2F2 in ovarian cancer and point to a unique scenario in which a single nuclear receptor plays potentially distinct roles in the stromal and epithelial compartments of the same tissue. PMID:23690307

Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis.

PubMed

Kugler, Jamie E; Passamaneck, Yale J; Feldman, Taya G; Beh, Jeni; Regnier, Todd W; Di Gregorio, Anna

2008-11-01

To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription. Copyright 2008 Wiley-Liss, Inc.

Treatment of collagenase-induced osteoarthritis with a viral vector encoding TSG-6 results in ectopic bone formation.

PubMed

Broeren, Mathijs G A; Di Ceglie, Irene; Bennink, Miranda B; van Lent, Peter L E M; van den Berg, Wim B; Koenders, Marije I; Blaney Davidson, Esmeralda N; van der Kraan, Peter M; van de Loo, Fons A J

2018-01-01

Tumor necrosis factor-inducible gene 6 (TSG-6) has anti-inflammatory and chondroprotective effects in mouse models of inflammatory arthritis. Because cartilage damage and inflammation are also observed in osteoarthritis (OA), we determined the effect of viral overexpression of TSG-6 in experimental osteoarthritis. Bone marrow-derived cells were differentiated to multinucleated osteoclasts in the presence of recombinant TSG-6 or after transduction with a lentiviral TSG-6 expression vector. Multi-nucleated osteoclasts were analyzed after tartrate resistant acid phosphatase staining and resorption activity was determined on dentin slices. Collagenase-induced osteoarthritis (CIOA) was induced in C57BL/6 mice after intra-articular injection of an adenoviral TSG-6 or control luciferase expression vector. Inflammation-related protease activity was measured using bioluminescent Prosense probes. After a second adenovirus injection, cartilage damage was assessed in histological sections stained with Safranin-O. Ectopic bone formation was scored in X-ray images of the affected knees. TSG-6 did not inhibit the formation of multi-nucleated osteoclasts, but caused a significant reduction in the resorption activity on dentin slices. Adenoviral TSG-6 gene therapy in CIOA could not reduce the cartilage damage compared to the luciferase control virus and no significant difference in inflammation-related protease activity was noted between the TSG-6 and control treated group. Instead, X-ray analysis and histological analysis revealed the presence of ectopic bone formation in the TSG-6 treated group. Gene therapy based on the expression of TSG-6 could not provide cartilage protection in experimental osteoarthritis, but instead resulted in increased ectopic bone formation.

Analysis of Expression Pattern and Genetic Deletion of Netrin5 in the Developing Mouse

PubMed Central

Garrett, Andrew M.; Jucius, Thomas J.; Sigaud, Liam P. R.; Tang, Fu-Lei; Xiong, Wen-Cheng; Ackerman, Susan L.; Burgess, Robert W.

2016-01-01

Boundary cap cells (BCC) are a transient, neural-crest-derived population found at the motor exit point (MEP) and dorsal root entry zone (DREZ) of the embryonic spinal cord. These cells contribute to the central/peripheral nervous system (CNS/PNS) boundary, and in their absence neurons and glia from the CNS migrate into the PNS. We found Netrin5 (Ntn5), a previously unstudied member of the netrin gene family, to be robustly expressed in BCC. We generated Ntn5 knockout mice and examined neurodevelopmental and BCC-related phenotypes. No abnormalities in cranial nerve guidance, dorsal root organization, or sensory projections were found. However, Ntn5 mutant embryos did have ectopic motor neurons (MNs) that migrated out of the ventral horn and into the motor roots. Previous studies have implicated semaphorin6A (Sema6A) in BCC signaling to plexinA2 (PlxnA2)/neuropilin2 (Nrp2) in MNs in restricting MN cell bodies to the ventral horn, particularly in the caudal spinal cord. In Ntn5 mutants, ectopic MNs are likely to be a different population, as more ectopias were found rostrally. Furthermore, ectopic MNs in Ntn5 mutants were not immunoreactive for NRP2. The netrin receptor deleted in colorectal cancer (DCC) is a potential receptor for NTN5 in MNs, as similar ectopic neurons were found in Dcc mutant mice, but not in mice deficient for other netrin receptors. Thus, Ntn5 is a novel netrin family member that is expressed in BCC, functioning to prevent MN migration out of the CNS. PMID:26858598

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

WEREWOLF and ENHANCER of GLABRA3 are interdependent regulators of the spatial expression pattern of GLABRA2 in Arabidopsis.

PubMed

Song, Sang-Kee; Kwak, Su-Hwan; Chang, Soo Chul; Schiefelbein, John; Lee, Myeong Min

2015-11-06

In multicellular organisms, cell fates are specified through differential regulation of transcription. Epidermal cell fates in the Arabidopsis thaliana root are precisely specified by several transcription factors, with the GLABRA2 (GL2) homeodomain protein acting at the farthest downstream in this process. To better understand the regulation of GL2 expression, we ectopically expressed WEREWOLF (WER) and ENHANCER OF GLABRA3 (EGL3) in various tissues and examined GL2 expression. Here we show that WER expressed ubiquitously in the root induced GL2 expression only in the root epidermis, whereas co-expression of WER and EGL3 induced GL2 expression in the corresponding tissues. We also found that GL3 accumulated in the nucleus at the early meristematic region and EGL3 accumulated later in the nucleus of epidermal cells. We further found that ectopic expression of WER and EGL3 in ground tissues inhibited GL2 expression in the epidermis. Our results suggest that the co-expression of WER and EGL3 is sufficient for driving GL2 and CPC expression. Copyright © 2015 Elsevier Inc. All rights reserved.

Curcumin-induced Aurora-A suppression not only causes mitotic defect and cell cycle arrest but also alters chemosensitivity to anticancer drugs.

PubMed

Ke, Ching-Shiun; Liu, Hsiao-Sheng; Yen, Cheng-Hsin; Huang, Guan-Cheng; Cheng, Hung-Chi; Huang, Chi-Ying F; Su, Chun-Li

2014-05-01

Overexpression of oncoprotein Aurora-A increases drug resistance and promotes lung metastasis of breast cancer cells. Curcumin is an active anticancer compound in turmeric and curry. Here we observed that Aurora-A protein and kinase activity were reduced in curcumin-treated human breast chemoresistant nonmetastatic MCF-7 and highly metastatic cancer MDA-MB-231 cells. Curcumin acts in a similar manner to Aurora-A small interfering RNA (siRNA), resulting in monopolar spindle formation, S and G2/M arrest, and cell division reduction. Ectopic Aurora-A extinguished the curcumin effects. The anticancer effects of curcumin were enhanced by Aurora-A siRNA and produced additivity and synergism effects in cell division and monopolar phenotype, respectively. Combination treatment with curcumin overrode the chemoresistance to four Food and Drug Administration (FDA)-approved anticancer drugs (ixabepilone, cisplatin, vinorelbine, or everolimus) in MDA-MB-231 cells, which was characterized by a decrease in cell viability and the occurrence of an additivity or synergy effect. Ectopic expression of Aurora-A attenuated curcumin-enhanced chemosensitivity to these four tested drugs. A similar benefit of curcumin was observed in MCF-7 cells treated with ixabepilone, the primary systemic therapy to patients with invasive breast cancer (stages IIA-IIIB) before surgery. Antagonism effect was observed when MCF-7 cells were treated with curcumin plus cisplatin, vinorelbine or everolimus. Curcumin-induced enhancement in chemosensitivity was paralleled by significant increases (additivity or synergy effect) in apoptosis and cell cycle arrest at S and G2/M phases, the consequences of Aurora-A inhibition. These results suggest that a combination of curcumin with FDA-approved anticancer drugs warrants further assessment with a view to developing a novel clinical treatment for breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of DDC-4/sFRP-4, a secreted frizzled-related protein, at the onset of apoptosis in mammary involution.

PubMed

Lacher, M D; Siegenthaler, A; Jäger, R; Yan, Xi; Hett, S; Xuan, L; Saurer, S; Lareu, R R; Dharmarajan, A M; Friis, R

2003-05-01

Using differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3beta (GSK-3beta), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.

MiR-24 alleviates cardiomyocyte apoptosis after myocardial infarction via targeting BIM.

PubMed

Pan, L-J; Wang, X; Ling, Y; Gong, H

2017-07-01

Ischemia hypoxia induces cardiomyocyte (CM) apoptosis in the process of acute myocardial infarction (AMI). It was showed that pro-apoptosis factor BIM participates in regulating tumor cell apoptosis under ischemia or hypoxia condition, while its role in CM apoptosis after AMI is still unclear. It was revealed that miR-24 expression was significantly reduced in myocardial tissue after AMI. Bioinformatics analysis exhibits that miR-24 is targeted to the 3'-UTR of BIM. This study aims to investigate the role of miR-24 in mediating BIM expression and CM apoptosis. Dual-luciferase assay was used to confirm the targeted regulation between miR-24 and BIM. Cells were cultured under ischemia hypoxia for 12 h after transfection for 48 h. Cell apoptosis was tested by using flow cytometry. The caspase activity was detected by using spectrophotometry. Wistar rats were divided into four groups, including Sham, AMI, AMI + agomir-control, and AMI + agomir-24 groups. Cardiac function was evaluated by using echocardiography. CM apoptosis was determined by using TUNEL. Infarction area was measured by using evans blue staining. MiR-24 targeted suppressed BIM expression. MiR-24 mimic and/or si-BIM transfection significantly declined the BIM expression, inhibited caspase-9 and caspase-3 activities, and reduced cell apoptosis in H9C2 cells. MiR-24 expression was decreased, while BIM levels were up-regulated in myocardium after AMI. Agomir-24 injection down-regulated the BIM expression in myocardium, reduced CM apoptosis, narrowed infarction area, and improved cardiac function in rats. MiR-24 was reduced, whereas BIM was enhanced in the CM after AMI. MiR-24 up-regulation plays a critical role in decreasing BIM expression, reducing CM apoptosis, and improving cardiac function after AMI.

Molecular analysis of the mouse agouti gene and the role of dominant agouti-locus mutations in obesity and insulin resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klebig, M.L.; Woychik, R.P.; Wilkinson, J.E.

1994-09-01

The lethal yellow (A{sup y/-}) and viable yellow (A{sup vy/-}) mouse agouti mutants have a predominantly yellow pelage and display a complex syndrome that includes obesity, hyperinsulinemia, and insulin resistance, hallmark features of obesity-associated noninsulin-dependent diabetes mellitus (NIDDM) in humans. A new dominant agouti allele, A{sup iapy}, has recently been identified; like the A{sup vy} allele, it is homozygous viable and confers obesity and yellow fur in heterozygotes. The agouti gene was cloned and characterized at the molecular level. The gene is expressed in the skin during hair growth and is predicted to encode a 131 amino acid protein, thatmore » is likely to be a secreted factor. In both Ay/- and A{sup iapy}/- mice, the obesity and other dominant pleiotropic effects are associated with an ectopic expression of agouti in many tissues where the gene product is normally not produced. In Ay, a 170-kb deletion has occurred that causes an upstream promoter to drive the ectopic expression of the wild-type agouti coding exons. In A{sup iapy}, the coding region of the gene is expressed from a cryptic promoter within the LTR of an intracisternal A-particle (IAP), which has integrated within the region just upstream of the first agouti coding exon. Transgenic mice ubiquitously expressing the cloned agouti gene under the influence of the beta-actin and phosphoglycerate kinase promoters display obesity, hyperinsulinemia, and yellow coat color. This demonstrates unequivocally that ectopic expression of agouti is responsible for the yellow obese syndrome.« less

PRDM1 expression on the epithelial component but not on ectopic lymphoid tissues of Warthin tumour.

PubMed

Wang, Y; Zhou, J; Zhang, Y; Wang, L; Liu, Y; Fan, L; Zhu, J; Xu, X; Huang, G; Li, X; Xun, W

2015-05-01

To determine the role of PRDM1, a key molecule for modulating the immune cells, in Warthin tumour (WT) pathogenesis. Forty paraffin-embedded parotid tissues of patients (mean age: 62.08 ± 11.90) with WT were retrieved from the pathology archives of Qindu Hospital from January 2012 to December 2012. The PRDM1 expression was investigated in a cohort of WT by immunohistochemistry. PRDM1 was expressed only on the epithelial component but not on ectopic lymphoid tissue of the tumour. Statistically, PRDM1 expression rates between WT glandular epithelial cells (40/40 cases) and the tumour-adjacent tissues (0/9 cases), and WT germinal centres (0/34 cases) and tonsil tissues (10/10 cases) were significantly different (P < 0.001), respectively. The PRDM1 expression appeared to play an essential role in WT pathogenesis. A better understanding of it might give options for revealing possible novel management strategies. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Efficient generation of induced pluripotent stem cells from human bone marrow mesenchymal stem cells.

PubMed

Yulin, X; Lizhen, L; Lifei, Z; Shan, F; Ru, L; Kaimin, H; Huang, H

2012-01-01

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression.

PubMed

Gong, Wei; Russell, Michael; Suzuki, Keiko; Riabowol, Karl

2006-04-01

ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.

Docetaxel-induced prostate cancer cell death involves concomitant activation of caspase and lysosomal pathways and is attenuated by LEDGF/p75

PubMed Central

Mediavilla-Varela, Melanie; Pacheco, Fabio J; Almaguel, Frankis; Perez, Jossymar; Sahakian, Eva; Daniels, Tracy R; Leoh, Lai Sum; Padilla, Amelia; Wall, Nathan R; Lilly, Michael B; De Leon, Marino; Casiano, Carlos A

2009-01-01

Background Hormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality. Results We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity. Conclusion These results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC. PMID:19715609

Characterization of the Olfactory Receptor OR10H1 in Human Urinary Bladder Cancer.

PubMed

Weber, Lea; Schulz, Wolfgang A; Philippou, Stathis; Eckardt, Josephine; Ubrig, Burkhard; Hoffmann, Michéle J; Tannapfel, Andrea; Kalbe, Benjamin; Gisselmann, Günter; Hatt, Hanns

2018-01-01

Olfactory receptors (ORs) are a large group of G-protein coupled receptors predominantly found in the olfactory epithelium. Many ORs are, however, ectopically expressed in other tissues and involved in several diseases including cancer. In this study, we describe that one OR, OR10H1, is predominantly expressed in the human urinary bladder with a notably higher expression at mRNA and protein level in bladder cancer tissues. Interestingly, also significantly higher amounts of OR10H1 transcripts were detectable in the urine of bladder cancer patients than in the urine of control persons. We identified the sandalwood-related compound Sandranol as a specific agonist of OR10H1. This deorphanization allowed the functional characterization of OR10H1 in BFTC905 bladder cancer cells. The effect of receptor activation was morphologically apparent in cell rounding, accompanied by changes in the cytoskeleton detected by β-actin, T-cadherin and β-Catenin staining. In addition, Sandranol treatment significantly diminished cell viability, cell proliferation and migration and induced a limited degree of apoptosis. Cell cycle analysis revealed an increased G1 fraction. In a concentration-dependent manner, Sandranol application elevated cAMP levels, which was reduced by inhibition of adenylyl cyclase, and elicited intracellular Ca 2+ concentration increase. Furthermore, activation of OR10H1 enhanced secretion of ATP and serotonin. Our results suggest OR10H1 as a potential biomarker and therapeutic target for bladder cancer.

miR-137 inhibits renal cell carcinoma growth in vitro and in vivo

PubMed Central

ZHANG, HONGXIA; LI, HONGJUN

2016-01-01

MicroRNA (miR)-137 has been reported to be underexpressed and involved in various cell processes and to have antitumor effects in a range of tumors, but so far not in renal cell carcinoma (RCC). The aim of the present study was to investigate the clinical significance and role of miR-137 in RCC. The expression levels of miR-137 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in 50 cases of paired RCC tissues and adjacent normal tissues, and in RCC cell lines. The role of miR-137 in the growth and survival of RCC cells was assessed with several in vitro approaches and in nude mice models. The results of the RT-qPCR showed that the miR-137 expression was downregulated in the RCC tissues and cell lines. The in vitro assay showed that ectopic expression of miR-137 robustly impaired RCC cell proliferation, migratory and invasive properties, and increased the induction of cell apoptosis properties. The in vivo assay demonstrated that enforced miR-137 suppressed tumor growth in xenograft nude mice models. In addition, miR-137 was indicated to inhibit the activation of phosphoinositide 3 kinase/protein kinase B signal pathway, which contributes to the inhibition of RCC growth. These findings indicate that miR-137 functions as tumor suppressor in RCC, suggesting that miR-137 may be a potential therapeutic target for RCC. PMID:27347205

MicroRNA-101-3p suppresses cell proliferation, invasion and enhances chemotherapeutic sensitivity in salivary gland adenoid cystic carcinoma by targeting Pim-1

PubMed Central

Liu, Xiao-Yu; Liu, Zhi-Jian; He, Hong; Zhang, Chen; Wang, Yun-Long

2015-01-01

MicroRNAs (miRNAs) play critical roles in carcinogenesis and tumor progression. Recent research has revealed miR-101-3p as an important regulator in several cancers. Nevertheless, its function in salivary gland Adenoid cystic carcinoma (ACC), a relatively rare malignance with poor long-term survival rate arisen in head and neck region, remain unknown. In this study, down-regulated miR-101-3p expression was detected in ACC tissues and ACC cell lines with high potential for metastasis. Ectopic expression of miR-101-3p significantly repressed the invasion, proliferation, colony formation, and formation of nude mice xenografts and induced potent apoptosis in ACC cell lines. The provirus integration site for Moloney murine leukemia virus 1 (Pim-1) oncogene was subsequently confirmed as a direct target gene of miR-101-3p in ACC. Functional restoration assays revealed that miR-101-3p inhibits cell growth and invasion by directly decreasing Pim-1 expression. Protein levels of Survivin, Cyclin D1 and β-catenin were also down-regulated by miR-101-3p. miR-101-3p enhanced the sensitivity of cisplatin in ACC cell lines. Taken together, our results demonstrate that the novel miR-101-3p/Pim-1 axis provides excellent insights into the carcinogenesis and tumor progression of ACC and may be a promising therapeutic target for this type of cancer. PMID:26693056

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.

PubMed

Tapia, Olga; Gerace, Larry

2016-01-01

We describe straightforward methodology for structure-function mapping of nuclear lamina proteins in myoblast differentiation, using populations of C2C12 myoblasts in which the endogenous lamina components are replaced with ectopically expressed mutant versions of the proteins. The procedure involves bulk isolation of C2C12 cell populations expressing the ectopic proteins by lentiviral transduction, followed by depletion of the endogenous proteins using siRNA, and incubation of cells under myoblast differentiation conditions. Similar methodology may be applied to mouse embryo fibroblasts or to other cell types as well, for the identification and characterization of sequences of lamina proteins involved in functions that can be measured biochemically or cytologically.

Ectopic bone formation in nude rats using human osteoblasts seeded poly(3)hydroxybutyrate embroidery and hydroxyapatite-collagen tapes constructs.

PubMed

Mai, Ronald; Hagedorn, Manolo Gunnar; Gelinsky, Michael; Werner, Carsten; Turhani, Dritan; Späth, Heike; Gedrange, Tomas; Lauer, Günter

2006-09-01

The aim of this study was to evaluate the ectopic bone formation using tissue engineered cell-seeded constructs with two different scaffolds and primary human maxillary osteoblasts in nude rats over an implantation period of up to 96 days. Collagen I-coated Poly(3)hydroxybutyrate (PHB) embroidery and hydroxyapatite (HAP) collagen tapes were seeded with primary human maxillary osteoblasts (hOB) and implanted into athymic rnu/run rats. A total of 72 implants were placed into the back muscles of 18 rats. 24, 48 and 96 days after implantation, histological and histomorphometric analyses were made. The osteoblastic character of the cells was confirmed by immunocytochemistry and RT-PCR for osteocalcin. Histological analysis demonstrated that all cell-seeded constructs induced ectopic bone formation after 24, 48 and 96 days of implantation. There was more mineralized tissue in PHB constructs than in HAP-collagen tapes (at day 24; p < 0.05). Bone formation decreased with the increasing length of the implantation period. Osteocalcin expression verified the osteoblastic character of the cell-seeded constructs after implantation time. No bone formation and no osteocalcin expression were found in the control groups. Cell-seeded constructs either with PHB embroidery or HAP-collagen tapes can induce ectopic bone formation. However, the amount of bone formed decreased with increasing length of implantation.

MACC1 regulates Fas mediated apoptosis through STAT1/3 - Mcl-1 signaling in solid cancers.

PubMed

Radhakrishnan, Harikrishnan; Ilm, Katharina; Walther, Wolfgang; Shirasawa, Senji; Sasazuki, Takehiko; Daniel, Peter T; Gillissen, Bernhard; Stein, Ulrike

2017-09-10

MACC1 was identified as a novel player in cancer progression and metastasis, but its role in death receptor-mediated apoptosis is still unexplored. We show that MACC1 knockdown sensitizes cancer cells to death receptor-mediated apoptosis. For the first time, we provide evidence for STAT signaling as a MACC1 target. MACC1 knockdown drastically reduced STAT1/3 activating phosphorylation, thereby regulating the expression of its apoptosis targets Mcl-1 and Fas. STAT signaling inhibition by the JAK1/2 inhibitor ruxolitinib mimicked MACC1 knockdown-mediated molecular signatures and apoptosis sensitization to Fas activation. Despite the increased Fas expression, the reduced Mcl-1 expression was instrumental in apoptosis sensitization. This reduced Mcl-1-mediated apoptosis sensitization was Bax and Bak dependent. MACC1 knockdown also increased TRAIL-induced apoptosis. MACC1 overexpression enhanced STAT1/3 phosphorylation and increased Mcl-1 expression, which was abrogated by ruxolitinib. The central role of Mcl-1 was strengthened by the resistance of Mcl-1 overexpressing cells to apoptosis induction. The clinical relevance of Mcl-1 regulation by MACC1 was supported by their positive expression correlation in patient-derived tumors. Altogether, we reveal a novel death receptor-mediated apoptosis regulatory mechanism by MACC1 in solid cancers through modulation of the STAT1/3-Mcl-1 axis. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of celecoxib on cell apoptosis and Fas, FasL and Bcl-2 expression in a BGC-823 human gastric cancer cell line.

PubMed

Li, Qian; Peng, Jie; Liu, Ting; Zhang, Guiying

2017-09-01

Fas, which is an apoptotic-related protein, has an important role in cell apoptosis. Fas ligand (FasL) binds to Fas and activates apoptosis signal transduction. We previously demonstrated that the efficiency of celecoxib inhibited the proliferation and apoptosis of HT-29 colon cancer cell line. The BGC823 cell line was used as an experimental model to evaluate the potential role of celecoxib on gastric cancer cell apoptosis. Inhibitory effects of celecoxib on cell viability were determined by MTT assay. Cell apoptosis was evaluated by flow cytometric analysis and laser confocal microscopy. The results of the present study demonstrated that celecoxib inhibited the viability of BGC823 cells in a concentration- and time-dependent manner. Furthermore, the effect of BGC823 cells apoptosis was increased in a concentration-dependent manner. Western blotting was used to determine the protein expression levels of Fas, FasL, and B-cell lymphoma-2 (Bcl-2). During the celecoxib-induced apoptosis of BGC823 cells, celecoxib upregulated Fas expression and downregulated FasL and Bcl-2 expression in a concentration-dependent manner. These results suggest that celecoxib inhibited the growth and induced apoptosis of BGC823 gastric cancer cells by regulating the protein expression of Fas, FasL and Bcl-2.

MiR-let-7a inhibits cell proliferation, migration, and invasion by down-regulating PKM2 in cervical cancer

PubMed Central

Guo, Man; Zhao, Xinying; Yuan, Xiaolei; Jiang, Jing; Li, Peiling

2017-01-01

In recent decades, miRNA has been reported as a crucial modulator in some biology progressions. This work aims to assess the expression and role of miR-let-7a and pyruvate kinase muscle isozyme M2 (PKM2) in CC tissues and cell lines. Here, we identified that miR-let-7a expression was decreased in CC tissues, and SiHa and HeLa cells (all P < 0.001), however, PKM2 expression was increased in these samples. Statistically, miR-let-7a was inversely associated with PKM2 mRNA or protein (p = 0.013, p = 0.015, respectively). In-vitro assays revealed that ectopic miR-let-7a expression repressed SiHa and HeLa cell proliferation, migration and invasion, and enhanced SiHa and HeLa cell apoptosis. Furthermore, luciferase reporter assays revealed the 3′-UTR of PKM2 was identified a target of miR-let-7a, by which miR-let-7a affected the expression of PKM2 in SiHa and HeLa cells. Besides, PKM2 plasmids partially abrogated the inhibitory effects of miR-let-7a, while si-PKM2 enhanced the inhibitory effects of miR-let-7a. In vivo, miR-let-7a mimics indeed repressed tumor growth in mice xenograft model. In conclusion, our results demonstrated that miR-let-7a inhibits cell proliferation, migration and invasion by down-regulation of PKM2 in cervical cancer. miR-let-7a/PKM2 pathway may be a useful therapeutic target for CC patients. PMID:28415668

Zebrafish pit1 mutants lack three pituitary cell types and develop severe dwarfism.

PubMed

Nica, Gabriela; Herzog, Wiebke; Sonntag, Carmen; Hammerschmidt, Matthias

2004-05-01

The Pou domain transcription factor Pit-1 is required for lineage determination and cellular commitment processes during mammalian adenohypophysis development. Here we report the cloning and mutational analysis of a pit1 homolog from zebrafish. Compared with mouse, zebrafish pit1 starts to be expressed at a much earlier stage of adenohypophysis development. However, as in the mouse, expression is restricted to a subset of pituitary cell types, excluding proopiomelanocortin (pomc)-expressing cells (corticotropes, melanotropes) and possibly gonadotropes. We could identify two N-ethyl-N-nitrosourea-induced zebrafish pit1 null mutants. Most mutants die during larval stages, whereas survivors develop severe dwarfism. Mutant larvae lack lactotropes, somatotropes, and thyrotropes, although the adenohypophysis is of normal size, without any sign of increased apoptosis rates. Instead, mutant embryos initiate ectopic expression of pomc in pit1-positive cells, leading to an expansion of the Pomc lineage. Similarly, the number of gonadotropes seems increased, as indicated by the expression of gsualpha, a marker for thyrotropes and gonadotropes. In pit1 mutants, the total number of gsualpha-positive cells is normal despite the loss of gsualpha and tshbeta coexpressing cells. Together, these data suggest a transfating of the Pit1 lineage to the Pomc and possibly the gonadotroph lineages in the mutant, and a pomc- and gonadotropin-repressive role of Pit1 during normal zebrafish development. This is different from mouse, for which a repressive role of Pit-1 has only been reported for the gonadotropin Lhbeta, but not for Pomc. In sum, our data point to both conserved and class-specific aspects of Pit1 function during pituitary development in different vertebrate species.

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.

Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibitedmore » the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.« less

MicroRNAs expression in ox-LDL treated HUVECs: MiR-365 modulates apoptosis and Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, Bing; Xiao, Bo; Liang, Desheng

Highlights: {yields} We evaluated the role of miRNAs in ox-LDL induced apoptosis in ECs. {yields} We found 4 up-regulated and 11 down-regulated miRNAs in apoptotic ECs. {yields} Target genes of the dysregulated miRNAs regulate ECs apoptosis and atherosclerosis. {yields} MiR-365 promotes ECs apoptosis via suppressing Bcl-2 expression. {yields} MiR-365 inhibitor alleviates ECs apoptosis induced by ox-LDL. -- Abstract: Endothelial cells (ECs) apoptosis induced by oxidized low-density lipoprotein (ox-LDL) is thought to play a critical role in atherosclerosis. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the expression of genes involved in diverse cell functions, including differentiation, growth,more » proliferation, and apoptosis. However, whether miRNAs are associated with ox-LDL induced apoptosis and their effect on ECs is still unknown. Therefore, this study evaluated potential miRNAs and their involvement in ECs apoptosis in response to ox-LDL stimulation. Microarray and qRT-PCR analysis performed on human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL identified 15 differentially expressed (4 up- and 11 down-regulated) miRNAs. Web-based query tools were utilized to predict the target genes of the differentially expressed miRNAs, and the potential target genes were classified into different function categories with the gene ontology (GO) term and KEGG pathway annotation. In particular, bioinformatics analysis suggested that anti-apoptotic protein B-cell CLL/lymphoma 2 (Bcl-2) is a target gene of miR-365, an apoptomir up-regulated by ox-LDL stimulation in HUVECs. We further showed that transfection of miR-365 inhibitor partly restored Bcl-2 expression at both mRNA and protein levels, leading to a reduction of ox-LDL-mediated apoptosis in HUVECs. Taken together, our findings indicate that miRNAs participate in ox-LDL-mediated apoptosis in HUVECs. MiR-365 potentiates ox-LDL-induced ECs apoptosis by regulating the expression of Bcl-2, suggesting potential novel therapeutic targets for atherosclerosis.« less

Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Lihua; Wang, Wensheng; Xiao, Weidong

2012-08-10

Highlights: Black-Right-Pointing-Pointer Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. Black-Right-Pointing-Pointer The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. Black-Right-Pointing-Pointer GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. Black-Right-Pointing-Pointer GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. Inmore » the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.« less

[Suppression of COX-2 protein to cell apoptosis in non-small cell lung cancer].

PubMed

Sun, Limei; Zhao, Yue; Wang, Lujian; Song, Min; Song, Jiye

2007-06-20

One of mechanisms of carcinogenesis is suppression of cell apoptosis which leads to accumulation of aberrant cells. The aim of this study is to investigate cell apoptosis and COX-2 protein expression in non-small cell lung cancer (NSCLC). Cell apoptosis, expression of COX-2 and microvessel density (MVD) were detcted in 111 NSCLC samples by TdT-mediated dUTP nick end labeling (TUNEL) technique and immunohistochemical staining. The positive rate of COX-2 protein expression was 67.6% (75/111), and there were 53 patients with high level cell apoptosis (47.7%). Expression of COX-2 protien was significantly related to TNM stages (P=0.025) and lymph node metastasis (P=0.018). The MVD in NSCLC tissues with positive COX-2 expression was significantly higher than that in negative expression ones (P=0.000). COX model showed that lymph node metastasis (P=0.006) and positive expression of COX-2 protein (P=0.000) were independent prognostic factors of NSCLC. The expression of COX-2 protein may suppress cell apoptosis of tumor, and it may serve as a potential marker of prognosis for NSCLC.

Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells.

PubMed

Min, Kyoung-Jin; Nam, Ju-Ock; Kwon, Taeg Kyu

2017-08-02

Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

Heterogeneous expression pattern of pro- and anti-apoptotic factors in myeloid progenitor cells of patients with severe congenital neutropenia treated with granulocyte colony-stimulating factor.

PubMed

Cario, Gunnar; Skokowa, Julia; Wang, Zheng; Bucan, Vesna; Zeidler, Cornelia; Stanulla, Martin; Schrappe, Martin; Welte, Karl

2005-04-01

Apoptosis is accelerated in the myeloid progenitor cells of patients with severe congenital neutropenia (CN). Granulocyte colony-stimulating factor (G-CSF) increases neutrophil numbers in most CN patients. The effect of G-CSF on apoptosis in CN was analysed by apoptosis rate and expression of anti- and pro-apoptotic factors. G-CSF-treated patients showed higher apoptosis frequency, lower expression of bcl-2 and bcl-xL, but higher expression of bfl-1/A1 and mcl-1. Caspase 9 was highly expressed in patients and controls after G-CSF administration. Thus, G-CSF acts on apoptosis regulation, but additional mechanisms leading to the increase of neutrophil numbers must be assumed.

Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis.

PubMed

Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

2017-05-01

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick‑end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers.

Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis

PubMed Central

Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

2017-01-01

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers. PMID:28447715

Coordination of Recombination with Meiotic Progression in the Caenorhabditis elegans Germline by KIN-18, a TAO Kinase That Regulates the Timing of MPK-1 Signaling

PubMed Central

Yin, Yizhi; Donlevy, Sean; Smolikove, Sarit

2016-01-01

Meiosis is a tightly regulated process requiring coordination of diverse events. A conserved ERK/MAPK-signaling cascade plays an essential role in the regulation of meiotic progression. The Thousand And One kinase (TAO) kinase is a MAPK kinase kinase, the meiotic role of which is unknown. We have analyzed the meiotic functions of KIN-18, the homolog of mammalian TAO kinases, in Caenorhabditis elegans. We found that KIN-18 is essential for normal meiotic progression; mutants exhibit accelerated meiotic recombination as detected both by analysis of recombination intermediates and by crossover outcome. In addition, ectopic germ-cell differentiation and enhanced levels of apoptosis were observed in kin-18 mutants. These defects correlate with ectopic activation of MPK-1 that includes premature, missing, and reoccurring MPK-1 activation. Late progression defects in kin-18 mutants are suppressed by inhibiting an upstream activator of MPK-1 signaling, KSR-2. However, the acceleration of recombination events observed in kin-18 mutants is largely MPK-1-independent. Our data suggest that KIN-18 coordinates meiotic progression by modulating the timing of MPK-1 activation and the progression of recombination events. The regulation of the timing of MPK-1 activation ensures the proper timing of apoptosis and is required for the formation of functional oocytes. Meiosis is a conserved process; thus, revealing that KIN-18 is a novel regulator of meiotic progression in C. elegans would help to elucidate TAO kinase’s role in germline development in higher eukaryotes. PMID:26510792

Infrasound exposure induces apoptosis of rat cardiac myocytes by regulating the expression of apoptosis-related proteins.

PubMed

Pei, Zhao-Hui; Chen, Bao-Ying; Tie, Ru; Zhang, Hai-Feng; Zhao, Ge; Qu, Ping; Zhu, Xiao-Xing; Zhu, Miao-Zhang; Yu, Jun

2011-12-01

It has been reported that exposure to infrasound causes cardiac dysfunction. Allowing for the key role of apoptosis in the pathogenesis of cardiovascular diseases, the objective of this study was to investigate the apoptotic effects of infrasound. Cardiac myocytes cultured from neonatal rats were exposed to infrasound of 5 Hz at 130 dB. The apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Also, the expression levels of a series of apoptosis-related proteins were detected. As a result, infrasound induced apoptosis of cultured rat cardiac myocytes in a time-dependant manner. The expression of proapoptotic proteins such as Bax, caspase-3, caspase-8, caspase-9, and FAS was significantly up-regulated, with concomitant down-regulated expression of antiapoptotic proteins such as Bcl-x, and the inhibitory apoptosis proteins family proteins including XIAP, cIAP-1, and cIAP-2. The expression of poly (ADP-ribose) polymerase and β-catenin, which are the substrate proteins of caspase-3, was significantly decreased. In conclusion, infrasound is an apoptotic inducer of cardiac myocytes.

The pro-apoptotic protein Bim is a convergence point for cAMP/protein kinase A- and glucocorticoid-promoted apoptosis of lymphoid cells.

PubMed

Zhang, Lingzhi; Insel, Paul A

2004-05-14

The mechanisms by which cAMP mediates apoptosis are not well understood. In the current studies, we used wild-type (WT) S49 T-lymphoma cells and the kin(-) variant (which lacks protein kinase A (PKA)) to examine cAMP/PKA-mediated apoptosis. The cAMP analog, 8-CPT-cAMP, increased phosphorylation of the cAMP response element-binding protein (CREB), activated caspase-3, and induced apoptosis in WT but not in kin(-) S49 cells. Using an array of 96 apoptosis-related genes, we found that treatment of WT cells with 8-CPT-cAMP for 24 h induced expression of mRNA for the pro-apoptotic gene, Bim. Real-time PCR analysis indicated that 8-CPT-cAMP increased Bim RNA in WT cells in <2 h and maintained this increase for >24 h. Bim protein expression increased in WT but not kin(-) cells treated with 8-CPT-cAMP or with the beta-adrenergic receptor agonist isoproterenol. Both apoptosis and Bim expression were reversible with removal of 8-CPT-cAMP after <6 h. The glucocorticoid dexamethasone also promoted apoptosis and Bim expression in S49 cells. In contrast, both UV light and anti-mouse Fas monoclonal antibody promoted apoptosis in S49 cells but did not induce Bim expression. 8-CPT-cAMP also induced Bim expression and enhanced dexamethasone-promoted apoptosis in human T-cell leukemia CEM-C7-14 (glucocorticoid-sensitive) and CEM-C1-15 (glucocorticoid-resistant) cells; increased Bim expression in 8-CPT-cAMP-treated CEM-C1-15 cells correlated with conversion of the cells from resistance to sensitivity to glucocorticoid-promoted apoptosis. Induction of Bim appears to be a key event in cAMP-promoted apoptosis in both murine and human T-cell lymphoma and leukemia cells and thus appears to be a convergence point for the killing of such cells by glucocorticoids and agents that elevate cAMP.

Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis).

PubMed

Zhou, Xiangjun; Fei, Zhangjun; Thannhauser, Theodore W; Li, Li

2011-11-23

Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5) was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant.

Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis)

PubMed Central

2011-01-01

Background Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Results Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5) was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. Conclusions The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant. PMID:22112144

A type III effector antagonises death receptor signalling during bacterial gut infection

PubMed Central

Pearson, Jaclyn S; Giogha, Cristina; Ong, Sze Ying; Kennedy, Catherine L; Kelly, Michelle; Robinson, Keith S; Wong, Tania; Mansell, Ashley; Riedmaier, Patrice; Oates, Clare VL; Zaid, Ali; Mühlen, Sabrina; Crepin, Valerie F; Marches, Olivier; Ang, Ching-Seng; Williamson, Nicholas A; O’Reilly, Lorraine A; Bankovacki, Aleksandra; Nachbur, Ueli; Infusini, Giuseppe; Webb, Andrew I; Silke, John; Strasser, Andreas; Frankel, Gad; Hartland, Elizabeth L

2013-01-01

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonise the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC), utilise a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonisation and interfere with antimicrobial host responses 1-3. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death domain containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death receptor induced apoptosis. This inhibition depended on the N-GlcNAc transferase activity of NleB1, which specifically modified Arg117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing (A/E) pathogens antagonise death receptor induced apoptosis of infected cells, thereby blocking a major antimicrobial host response. PMID:24025841

mRNA-binding protein TIA-1 reduces cytokine expression in human endometrial stromal cells and is down-regulated in ectopic endometrium.

PubMed

Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D; Kristiansson, Helena; Duke, Cindy M P; Choe, Gina; Flannery, Clare; Kallen, Caleb B; Seli, Emre

2014-12-01

Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3'-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Endometrial TIA-1 is regulated throughout the menstrual cycle, TIA-1 modulates the expression of immune factors in endometrial cells, and downregulation of TIA-1 may contribute to the pathogenesis of endometriosis.

mRNA-Binding Protein TIA-1 Reduces Cytokine Expression in Human Endometrial Stromal Cells and Is Down-Regulated in Ectopic Endometrium

PubMed Central

Karalok, Hakan Mete; Aydin, Ebru; Saglam, Ozlen; Torun, Aysenur; Guzeloglu-Kayisli, Ozlem; Lalioti, Maria D.; Kristiansson, Helena; Duke, Cindy M. P.; Choe, Gina; Flannery, Clare; Kallen, Caleb B.

2014-01-01

Background: Cytokines and growth factors play important roles in endometrial function and the pathogenesis of endometriosis. mRNAs encoding cytokines and growth factors undergo rapid turnover; primarily mediated by adenosine- and uridine-rich elements (AREs) located in their 3′-untranslated regions. T-cell intracellular antigen (TIA-1), an mRNA-binding protein, binds to AREs in target transcripts, leading to decreased gene expression. Objective: The purpose of this article was to determine whether TIA-1 plays a role in the regulation of endometrial cytokine and growth factor expression during the normal menstrual cycle and whether TIA-1 expression is altered in women with endometriosis. Methods: Eutopic endometrial tissue obtained from women without endometriosis (n = 30) and eutopic and ectopic endometrial tissues from women with endometriosis (n = 17) were immunostained for TIA-1. Staining intensities were evaluated by histological scores (HSCOREs). The regulation of endometrial TIA-1 expression by immune factors and steroid hormones was studied by treating primary cultured human endometrial stromal cells (HESCs) with vehicle, lipopolysaccharide, TNF-α, IL-6, estradiol, or progesterone, followed by protein blot analyses. HESCs were engineered to over- or underexpress TIA-1 to test whether TIA-1 regulates IL-6 or TNF-α expression in these cells. Results: We found that TIA-1 is expressed in endometrial stromal and glandular cells throughout the menstrual cycle and that this expression is significantly higher in the perimenstrual phase. In women with endometriosis, TIA-1 expression in eutopic and ectopic endometrium was reduced compared with TIA-1 expression in eutopic endometrium of unaffected control women. Lipopolysaccharide and TNF-α increased TIA-1 expression in HESCs in vitro, whereas IL-6 or steroid hormones had no effect. In HESCs, down-regulation of TIA-1 resulted in elevated IL-6 and TNF-α expression, whereas TIA-1 overexpression resulted in decreased IL-6 and TNF-α expression. Conclusions: Endometrial TIA-1 is regulated throughout the menstrual cycle, TIA-1 modulates the expression of immune factors in endometrial cells, and downregulation of TIA-1 may contribute to the pathogenesis of endometriosis. PMID:25140393

Small Interfering RNA-Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells.

PubMed

Park, Jong-Beom; Park, Chanjoo

2017-10-01

In vitro cell culture model. To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells. Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear. Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls. Serum deprivation increased apoptosis by 40.3% ( p <0.001), decreased proliferation by 45.3% ( p <0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures ( p <0.001). Finally, Fas siRNA-mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures ( p <0.05 for both). The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

Trichostatin A-induced apoptosis is mediated by Kruppel-like factor 4 in ovarian and lung cancer.

PubMed

Zohre, Sadeghi; Kazem, Nejati-Koshki; Abolfazl, Akbarzadeh; Mohammad, Rahmati-Yamchi; Aliakbar, Movassaghpour; Effat, Alizadeh; Zahra, Davoudi; Hassan, Dariushnejad; Nosratollah, Zarghami

2014-01-01

The istone deacetylase (HDAC) inhibitor trichostatin A (TSA) is known to mediate the regulation of gene expression and anti proliferation activity in cancer cells. Kruppel-like factor 4 (klf4) is a zinc finger- containing transcription factor of the SP/KLF family, that is expressed in a variety of tissues and regulates cell proliferation, differentiation, tumorigenesis, and apoptosis. It may either either function as a tumor suppressor or an oncogene depending on genetic context of tumors. In this study, we tested the possibility that TSA may increase klf4 expression and cancer cell growth inhibition and apoptosis in SKOV-3 and A549 cells. The cytotoxicity of TSA was determined using the MTT assay test, while klf4 gene expression was assessed by real time PCR and to ability of TSA to induce apoptosis using a Vybrant Apoptosis Assay kit. Our results showed that TSA exerted dose and time dependent cytotoxicity effect on SKOV-3 and A549 cells. Moreover TSA up-regulated klf4 expression. Flow cytometric analysis demonstrated that apoptosis was increased after TSA treatment. Taken together, this study showed that TSA increased klf4 expression in SKOV3 and A549 cell lines, consequently, klf4 may played a tumor-suppressor role by increasing both cell growth inhibition and apoptosis. This study sheds light on the details of molecular mechanisms of HDACI-induced cell cycle arrest and apoptosis.

Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium.

PubMed

Humby, Frances; Bombardieri, Michele; Manzo, Antonio; Kelly, Stephen; Blades, Mark C; Kirkham, Bruce; Spencer, Jo; Pitzalis, Costantino

2009-01-13

Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium. Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Igamma-Cmu circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis. Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell-depleting therapies.

FGFR1 inhibits skeletal muscle atrophy associated with hindlimb suspension

PubMed Central

Eash, John; Olsen, Aaron; Breur, Gert; Gerrard, Dave; Hannon, Kevin

2007-01-01

Background Skeletal muscle atrophy can occur under many different conditions, including prolonged disuse or immobilization, cachexia, cushingoid conditions, secondary to surgery, or with advanced age. The mechanisms by which unloading of muscle is sensed and translated into signals controlling tissue reduction remains a major question in the field of musculoskeletal research. While the fibroblast growth factors (FGFs) and their receptors are synthesized by, and intimately involved in, embryonic skeletal muscle growth and repair, their role maintaining adult muscle status has not been examined. Methods We examined the effects of ectopic expression of FGFR1 during disuse-mediated skeletal muscle atrophy, utilizing hindlimb suspension and DNA electroporation in mice. Results We found skeletal muscle FGF4 and FGFR1 mRNA expression to be modified by hind limb suspension,. In addition, we found FGFR1 protein localized in muscle fibers within atrophying mouse muscle which appeared to be resistant to atrophy. Electroporation and ectopic expression of FGFR1 significantly inhibited the decrease in muscle fiber area within skeletal muscles of mice undergoing suspension induced muscle atrophy. Ectopic FGFR1 expression in muscle also significantly stimulated protein synthesis in muscle fibers, and increased protein degradation in weight bearing muscle fibers. Conclusion These results support the theory that FGF signaling can play a role in regulation of postnatal skeletal muscle maintenance, and could offer potentially novel and efficient therapeutic options for attenuating muscle atrophy during aging, illness and spaceflight. PMID:17425786

Overexpressing circular RNA hsa_circ_0002052 impairs osteosarcoma progression via inhibiting Wnt/β-catenin pathway by regulating miR-1205/APC2 axis.

PubMed

Wu, Zhen; Shi, Wangping; Jiang, Chendi

2018-08-25

Circular RNAs (circRNAs) are a novel class of noncoding RNAs, whose importance in cancer has been gradually acknowledged. However, the functions of circRNAs in tumorigenesis have not been fully understood. In the present study, we identified a novel circRNA hsa_circ_0002052 significantly downregulated in osteosarcoma (OS) tissues and cell lines. Moreover, we found that hsa_circ_0002052 could act as a biomarker to indicate the prognosis of OS patients. Functionally, we showed that hsa_circ_0002052 overexpression significantly suppressed OS cell proliferation, migration and invasion while promoting apoptosis in vitro. Similarly, in vivo assay indicated that ectopic expression of hsa_circ_0002052 impaired OS cell growth. In terms of mechanism, we found that hsa_circ_0002052 inhibited miR-1205 while miR1205 targeted APC2, a negative regulator of Wnt/β-catenin signaling pathway. By releasing the inhibition of miR-1205 on APC2 expression, hsa_circ_0002052 suppressed the activation of Wnt/β-catenin signaling pathway, leading to attenuated OS progression. Taken together, our study for the first time revealed a suppressive circRNA hsa_circ_0002052 involved in OS progression. Our study suggested hsa_circ_0002052/miR-1205/APC2/Wnt/β-catenin axis might be a potential target for OS therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1.

PubMed

Majumder, Pritha; Chattopadhyay, Biswanath; Mazumder, Arindam; Das, Pradeep; Bhattacharyya, Nitai P

2006-05-01

To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

Spontaneous endometriosis in a mandrill (Mandrillus sphinx).

PubMed

Nakamura, S; Ochiai, K; Ochi, A; Ito, M; Kamiya, T; Yamamoto, H

2012-01-01

A 25-year-old female mandrill (Mandrillus sphinx) died after exhibiting weakness and recumbency with serosanguineous ascites. Gross findings included haemoperitoneum and multifocal to diffuse serosal thickening with petechiae and ecchymoses throughout the peritoneum. The uterus was covered entirely with large blood clots and was adherent to the ovaries and pelvic wall. Microscopical and immunohistochemical examination revealed extra- and intra-uterine growth of ectopic endometrial tissue with marked fibrosis. The ectopic endometrial tissues predominantly consisted of stromal cells expressing CD10 and progesterone receptor and variably-sized glands lined by the epithelium with occasional slight expression of oestrogen receptor α. A diagnosis of endometriosis was made. This is the first report of naturally occurring endometriosis in a mandrill. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant.

PubMed Central

Tsuchimoto, S; van der Krol, A R; Chua, N H

1993-01-01

We cloned a MADS-box gene, pMADS3, from Petunia hybrida, which shows high sequence homology to the Arabidopsis AGAMOUS and Antirrhinum PLENA. pMADS3 is expressed exclusively in stamens and carpels of wild-type petunia plants. In the petunia mutant blind, which shows homeotic conversions of corolla limbs into antheroid structures with pollen grains and small parts of sepals into carpelloid tissue, pMADS3 is expressed in all floral organs as well as in leaves. Ectopic expression of pMADS3 in transgenic petunia leads to phenocopies of the blind mutant, i.e., the formation of antheroid structures on limbs and carpelloid tissue on sepals. Transgenic tobacco plants that overexpress pMADS3 exhibit an even more severe phenotype, with the sepals forming a carpel-like structure encasing the interior floral organs. Our results identify BLIND as a negative regulator of pMADS3, which specifies stamens and carpels during petunia flower development. PMID:8104573

Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

PubMed

Fryer, J P; Oetting, W S; Brott, M J; King, R A

2001-11-01

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Tazarotene-Induced Gene 1 Interacts with DNAJC8 and Regulates Glycolysis in Cervical Cancer Cells.

PubMed

Wang, Chun-Hua; Shyu, Rong-Yaun; Wu, Chang-Chieh; Chen, Mao-Liang; Lee, Ming-Cheng; Lin, Yi-Yin; Wang, Lu-Kai; Jiang, Shun-Yuan; Tsai, Fu-Ming

2018-06-14

The tazarotene-induced gene 1 (TIG1) protein is a retinoidinducible growth regulator and is considered a tumor suppressor. Here, we show that DnaJ heat shock protein family member C8 (DNAJC8) is a TIG1 target that regulates glycolysis. Ectopic DNAJC8 expression induced the translocation of pyruvate kinase M2 (PKM2) into the nucleus, subsequently inducing glucose transporter 1 (GLUT1) expression to promote glucose uptake. Silencing either DNAJC8 or PKM2 alleviated the upregulation of GLUT1 expression and glucose uptake induced by ectopic DNAJC8 expression. TIG1 interacted with DNAJC8 in the cytosol, and this interaction completely blocked DNAJC8-mediated PKM2 translocation and inhibited glucose uptake. Furthermore, increased glycose uptake was observed in cells in which TIG1 was silenced. In conclusion, TIG1 acts as a pivotal repressor of DNAJC8 to enhance glucose uptake by partially regulating PKM2 translocation.

Long non-coding RNA HOTAIR promotes UVB-induced apoptosis and inflammatory injury by up-regulation of PKR in keratinocytes.

PubMed

Liu, Guo; Zhang, Wenhao

2018-06-11

Excessive exposure to ultraviolet (UV) rays can cause damage of the skin and may induce cancer, immunosuppression, photoaging, and inflammation. The long non-coding RNA (lncRNA) HOX antisense intergenic RNA (HOTAIR) is involved in multiple human biological processes. However, its role in UVB-induced keratinocyte injury is unclear. This study was performed to investigate the effects of HOTAIR in UVB-induced apoptosis and inflammatory injury in human keratinocytes (HaCaT cells). Quantitative real-time polymerase chain reaction was performed to analyze the expression levels of HOTAIR, PKR, TNF-α, and IL-6. Cell viability was measured using trypan blue exclusion method and cell apoptosis using flow cytometry and western blot. ELISA was used to measure the concentrations of TNF-α and IL-6. Western blot was used to measure the expression of PKR, apoptosis-related proteins, and PI3K/AKT and NF-κB pathway proteins. UVB induced HaCaT cell injury by inhibiting cell viability and promoting cell apoptosis and expressions of IL-6 and TNF-α. UVB also promoted the expression of HOTAIR. HOTAIR suppression increased cell viability and decreased apoptosis and expression of inflammatory factors in UVB-treated cells. HOTAIR also promoted the expression of PKR. Overexpression of HOTAIR decreased cell viability and increased cell apoptosis and expression of inflammatory factors in UVB-treated cells by upregulating PKR. Overexpression of PKR decreased cell viability and promoted cell apoptosis in UVB-treated cells. Overexpression of PKR activated PI3K/AKT and NF-κB pathways. Our findings identified an essential role of HOTAIR in promoting UVB-induced apoptosis and inflammatory injury by up-regulating PKR in keratinocytes.

Apigenin Induces the Apoptosis and Regulates MAPK Signaling Pathways in Mouse Macrophage ANA-1 Cells

PubMed Central

Liao, Yuexia; Shen, Weigan; Kong, Guimei; Lv, Houning; Tao, Wenhua; Bo, Ping

2014-01-01

Apigenin is a naturally occurring plant flavonoid that possesses antioxidant, anti-cancer and anti-inflammatory properties. However, there are few reports has been done on the ability of apigenin to induce apoptosis in macrophages. In this study, mouse macrophage ANA-1 cells were incubated with different concentrations of apigenin. The cell viability was determined by an MTT assay. The cell apoptosis were analyzed by flow cytometric analysis. Apoptosis were also analyzed using a TUNEL assay and a DNA ladder. The level of intracellular ROS was detected using a dichlorofluorescein -diacetate probe. The expression levels of apoptosis-related proteins were detected by western blot analysis. The results showed that apigenin decreased the viability of ANA-1 cells and induced apoptosis in a dose- and time-dependent manner. Apigenin increased the level of intracellular ROS, downregulated the expression of Bcl-2 and upregulated the expression of caspase-3 and caspase-8 in ANA-1 cells. Furthermore, apigenin downregulated the expression of phospho-ERK and phospho-JNK, upregulated the expression of phospho-p38 and had no significant effect on the expression of Bax, ERK, JNK and p38. The results suggested that apigenin induced cell apoptosis in mouse macrophage ANA-1 cells may via increasing intracellular ROS, regulating the MAPK pathway, and then inhibiting Bcl-2 expression. PMID:24646936

Augmented cell survival in eutopic endometrium from women with endometriosis: Expression of c-myc, TGF-beta1 and bax genes

PubMed Central

Johnson, M Cecilia; Torres, Marisa; Alves, Alessandra; Bacallao, Ketty; Fuentes, Ariel; Vega, Margarita; Boric, M Angélica

2005-01-01

Background Endometriosis is a common gynaecological disorder characterized by the presence of endometrial tissue outside of the uterus. The fragments in normal menstruation are composed of necrotic and living cells, which do not survive in ectopic locations because of programmed cell death. The aim of this study was to evaluate if the balance between cell proliferation and apoptosis is changed in eutopic endometrium from women with endometriosis throughout the menstrual cycle by studying bax (pro-apoptotic), c-myc (regulator of cell cycle) and TGF-beta1 (involved in cell differentiation) genes. Methods Eutopic endometrium was obtained from: 30 women with endometriosis (32.8 +/- 5 years) and 34 fertile eumenorrheic women (36 +/- 5.3 years). We analyzed apoptosis (TUNEL: DNA fragmentation); cell proliferation (immunohistochemistry (IHC) for Ki67); c-myc, bax and TGF-beta1 mRNA abundance (RT-PCR) and TGF-beta1 protein (IHC) in endometrial explants. Results Cell proliferation strongly decreased from proliferative to late secretory phases in glands, but not in stroma, in both endometria. Positive staining in glands and stroma from proliferative endometrium with endometriosis was 1.9- and 2.2-fold higher than control endometrium, respectively (p < 0.05). Abundance of c-myc mRNA was 65% higher in proliferative endometrium from endometriosis than normal tissue (p < 0.05). TGF-beta1 (mRNA and protein) augmented during mid secretory phase in normal endometrium, effect not observed in endometrium with endometriosis. In normal endometrium, the percentage of apoptotic epithelial and stromal cells increased more than 30-fold during late secretory phase. In contrast, in endometrium from endometriosis, not only this increase was not observed, besides bax mRNA decreased 63% versus normal endometrium (p < 0.05). At once, in early secretory phase, apoptotic stromal cells increased 10-fold with a concomitant augment of bax mRNA abundance (42%) in endometria from endometriosis (p < 0.05). Conclusion An altered expression of c-myc, TGF-beta1 and bax was observed in eutopic endometrium from endometriosis, suggesting its participation in the regulation of cell survival in this disease. The augmented cell viability in eutopic endometrium from these patients as a consequence of a reduction in cell death by apoptosis, and also an increase in cell proliferation indicates that this condition may facilitate the invasive feature of the endometrium. PMID:16150151

Cross-regulation in the mouse HoxB complex: the expression of Hoxb2 in rhombomere 4 is regulated by Hoxb1.

PubMed

Maconochie, M K; Nonchev, S; Studer, M; Chan, S K; Pöpperl, H; Sham, M H; Mann, R S; Krumlauf, R

1997-07-15

Correct regulation of the segment-restricted patterns of Hox gene expression is essential for proper patterning of the vertebrate hindbrain. We have examined the molecular basis of restricted expression of Hoxb2 in rhombomere 4 (r4), by using deletion analysis in transgenic mice to identify an r4 enhancer from the mouse gene. A bipartite Hox/Pbx binding motif is located within this enhancer, and in vitro DNA binding experiments showed that the vertebrate labial-related protein Hoxb1 will cooperatively bind to this site in a Pbx/Exd-dependent manner. The Hoxb2 r4 enhancer can be transactivated in vivo by the ectopic expression of Hoxb1, Hoxa1, and Drosophila labial in transgenic mice. In contrast, ectopic Hoxb2 and Hoxb4 are unable to induce expression, indicating that in vivo this enhancer preferentially responds to labial family members. Mutational analysis demonstrated that the bipartite Hox/Pbx motif is required for r4 enhancer activity and the responses to retinoids and ectopic Hox expression. Furthermore, three copies of the Hoxb2 motif are sufficient to mediate r4 expression in transgenic mouse embryos and a labial pattern in Drosophila embryos. This reporter expression in Drosophila embryos is dependent upon endogenous labial and exd, suggesting that the ability of this Hox/Pbx site to interact with labial-related proteins has been evolutionarily conserved. The endogenous Hoxb2 gene is no longer upregulated in r4 in Hoxb1 homozygous mutant embryos. On the basis of these experiments we conclude that the r4-restricted domain of Hoxb2 in the hindbrain is the result of a direct cross-regulatory interaction by Hoxb1 involving vertebrate Pbx proteins as cofactors. This suggests that part of the functional role of Hoxb1 in maintaining r4 identity may be mediated by the Hoxb2 gene.

Protein Kinase C- ɛ Regulates the Apoptosis and Survival of Glioma Cells

PubMed Central

Okhrimenko, Hana; Lu, Wei; Xiang, Cunli; Hamburger, Nathan; Kazimirsky, Gila; Brodie, Chaya

2005-01-01

In this study, we examined the role of protein kinase C (PKC)-ɛ in the apoptosis and survival of glioma cells using tumor necrosis factor–related apoptosis inducing ligand (TRAIL)- stimulated cells and silencing of PKCɛ expression. Treatment of glioma cells with TRAIL induced activation, caspase-dependent cleavage, and down-regulation of PKCɛ within 3 to 5 hours of treatment. Overexpression of PKCɛ inhibited the apoptosis induced by TRAIL, acting downstream of caspase 8 and upstream of Bid cleavage and cytochrome c release from the mitochondria. A caspase-resistant PKCɛ mutant (D383A) was more protective than PKCɛ, suggesting that both the cleavage of PKCɛ and its down-regulation contributed to the apoptotic effect of TRAIL. To further study the role of PKCɛ in glioma cell apoptosis, we employed short interfering RNAs directed against the mRNA of PKCɛ and found that silencing of PKCɛ expression induced apoptosis of various glioma cell lines and primary glioma cultures. To delineate the molecular mechanisms involved in the apoptosis induced by silencing of PKCɛ, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that knockdown of PKCɛ did not affect the expression of Bcl2 and Bax or the phosphorylation and expression of Erk1/2, c-Jun-NH2-kinase, p38, or STAT, whereas it selectively reduced the expression of AKT. Similarly, TRAIL reduced the expression of AKT in glioma cells and this decrease was abolished in cells overexpressing PKCɛ. Our results suggest that the cleavage of PKCɛ and its down-regulation play important roles in the apoptotic effect of TRAIL. Moreover, PKCɛ regulates AKT expression and is essential for the survival of glioma cells. PMID:16103081

Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium

PubMed Central

Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J.; Klein, Ophir D.; Barlow, Linda A.

2014-01-01

Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation. PMID:24993944

Meta-analysis of expression of l(3)mbt tumor-associated germline genes supports the model that a soma-to-germline transition is a hallmark of human cancers.

PubMed

Feichtinger, Julia; Larcombe, Lee; McFarlane, Ramsay J

2014-05-15

Evidence is starting to emerge indicating that tumorigenesis in metazoans involves a soma-to-germline transition, which may contribute to the acquisition of neoplastic characteristics. Here, we have meta-analyzed gene expression profiles of the human orthologs of Drosophila melanogaster germline genes that are ectopically expressed in l(3)mbt brain tumors using gene expression datasets derived from a large cohort of human tumors. We find these germline genes, some of which drive oncogenesis in D. melanogaster, are similarly ectopically activated in a wide range of human cancers. Some of these genes normally have expression restricted to the germline, making them of particular clinical interest. Importantly, these analyses provide additional support to the emerging model that proposes a soma-to-germline transition is a general hallmark of a wide range of human tumors. This has implications for our understanding of human oncogenesis and the development of new therapeutic and biomarker targets with clinical potential. © 2013 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

Regulatory role of AINTEGUMENTA in organ initiation and growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krizek, Beth Allyn; Lebioda, Lukasz

2005-03-01

Although several members of the plant-specific AP2/ERF family of transcription factors are important developmental regulators, many genes in this large protein family remain uncharacterized. Here, we present a phylogenetic analysis of the18 genes that make up the AP2 subgroup of this family. We report expression analyses of seven Arabidopsis genes most closely related to the floral development gene AINTEGUMENTA and show that all AINTEGUMENTA-like (AIL) genes are transcribed in multiple tissues during development. They are expressed primarily in young actively dividing tissues of a plant and not in mature leaves or stems. The spatial distribution of AIL5, AIL6, and AIL7more » mRNA in inflorescences was characterized by in situ hybridization. Each of these genes is expressed in a spatially and temporally distinct pattern within inflorescence meristems and flowers. Ectopic expression of AIL5 resulted in a larger floral organ phenotype, similar to that resulting from ectopic expression of ANT. Our results are consistent with AIL genes having roles in specification of meristematic or division-competent states.« less

Endometrial Expression of Steroidogenic Factor 1 Promotes Cystic Glandular Morphogenesis

PubMed Central

Vasquez, Yasmin M.; Wu, San-Pin; Anderson, Matthew L.; Hawkins, Shannon M.; Creighton, Chad J.; Ray, Madhumita; Tsai, Sophia Y.; Tsai, Ming-Jer; Lydon, John P.

2016-01-01

Epigenetic silencing of steroidogenic factor 1 (SF1) is lost in endometriosis, potentially contributing to de novo local steroidogenesis favoring inflammation and growth of ectopic endometrial tissue. In this study, we examine the impact of SF1 expression in the eutopic uterus by a novel mouse model that conditionally expresses SF1 in endometrium. In vivo SF1 expression promoted the development of enlarged endometrial glands and attenuated estrogen and progesterone responsiveness. Endometriosis induction by autotransplantation of uterine tissue to the mesenteric membrane resulted in the increase in size of ectopic lesions from SF1-expressing mice. By integrating the SF1-dependent transcriptome with the whole genome binding profile of SF1, we identified uterine-specific SF1-regulated genes involved in Wingless and Progesterone receptor-Hedgehog-Chicken ovalbumin upstream promoter transcription factor II signaling for gland development and epithelium-stroma interaction, respectively. The present results indicate that SF1 directly contributes to the abnormal uterine gland morphogenesis, an inhibition of steroid hormone signaling and activation of an immune response, in addition to previously postulated estrogen production. PMID:27018534

Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

PubMed

Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

2016-01-01

Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

[Apoptosis and expression of apoptosis-related proteins in experimental different denervated guinea-pig facial muscle].

PubMed

Hui, Lian; Wei, Hong-Quan; Li, Xiao-Tian; Guan, Chao; Ren, Zhong

2005-02-01

To study apoptosis and expression of apoptosis-related proteins in experimental different denervated guinea-pig facial muscle. An experimental model was established with guinea pigs by compressing the facial nerve 30 second (reinnervated group) and resecting the facial nerve (denervated group). TUNEL method and immunohistochemical technique (SABC) were applied to detect the apoptosis and expression of apoptosis-related proteins bcl-2 and bax from 1st to 8th week after operation. Experimentally denervated facial muscle revealed consistently increase of DNA fragmentation, average from(34.4 +/- 4.6)% to (38.2 +/- 10.6)%, from 1st week to 8th week after operation; Reinnervated facial muscle showed a temporal increase of DNA fragmentation, and then the muscle fiber nuclei revealed decreased DNA fragmentation along with the function of facial nerve recovered, latterly normal, average from (32.0 +/- 8.03)% to (5.6 +/- 3.5)%, from 1st week to 8th week after operation. In denervated group, bcl-2 and bax were expressed strongly; in reinnervated group, bcl-2 expressed consistently, but bax disappeared latterly along with the function of facial nerve recovered. Expression of DNA fragmentation and apoptosis-related proteins in denervated muscle are general reaction to denervation. bcl-2 can prevent early apoptotic muscle fiber to survival until reinnervation. It is concluded that proteins control apoptosis may give information for possible therapeutic interventions to reduce the rate of muscle fiber death in denervated atrophy in absence of effective primary treatment.

Bcl-2 protein expression associated with resistance to apoptosis in clear cell adenocarcinomas of the vagina and cervix expressing wild-type p53.

PubMed

Waggoner, S E; Baunoch, D A; Anderson, S A; Leigh, F; Zagaja, V G

1998-09-01

Clear cell adenocarcinomas (CCAs) of the vagina and cervix are rare tumors that often overexpress wild-type p53. In vitro, expression of protooncogene bcl-2 can block p53-mediated apoptosis. The objective of this study was to determine if bcl-2 is expressed in CCAs and whether this expression is associated with inhibition of apoptosis. Twenty-one paraffin-embedded clear cell adenocarcinomas were immunohistochemically stained for bcl-2 (antibody M 887, Dako, Carpinteria, CA) and DNA fragmentation (ApopTag, Oncor, Gaithersburg, MD), a marker for apoptosis. Fifteen tumors were associated with in utero exposure to diethylstilbestrol (DES). Prior p53 gene analysis had indicated the presence of wild-type p53 in each tumor. Human lymphoid tissue containing bcl-2-expressing lymphocytes and DNase I-exposed CCA tissue sections were used as positive controls for the bcl-2 and apoptosis assays, respectively. Expression of bcl-2 and DNA fragmentation was classified (0 to 3+) according to percentage of positive cells and intensity of staining. Expression of bcl-2 was identified in each CCA examined, and was strongly positive (2+ to 3+) in 18 of 21 samples. Despite the presence of wild-type p53, only 4 of 21 tumors showed evidence of apoptosis as assessed through DNA fragmentation. DNA damage leads to increased intracellular p53 levels. Overexpression of p53 induces apoptosis as a means of protecting organisms from the development of malignancy. CCAs of the vagina and cervix, which contain wild-type p53 genes and often overexpress p53 protein, presumably have evolved mechanisms to avoid p53-induced apoptosis. Our observations are consistent with the hypothesis that overexpression of bcl-2 can inhibit p53-mediated apoptosis and suggest a mechanism by which these rare tumors can arise without mutation of the p53 gene.

Bupivacaine-induced apoptosis independently of WDR35 expression in mouse neuroblastoma Neuro2a cells

PubMed Central

2012-01-01

Background Bupivacaine-induced neurotoxicity has been shown to occur through apoptosis. Recently, bupivacaine was shown to elicit reactive oxygen species (ROS) production and induce apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) in a human neuroblastoma cell line. We have reported that WDR35, a WD40-repeat protein, may mediate apoptosis through caspase-3 activation. The present study was undertaken to test whether bupivacaine induces apoptosis in mouse neuroblastoma Neuro2a cells and to determine whether ROS, p38 MAPK, and WDR35 are involved. Results Our results showed that bupivacaine induced ROS generation and p38 MAPK activation in Neuro2a cells, resulting in apoptosis. Bupivacaine also increased WDR35 expression in a dose- and time-dependent manner. Hydrogen peroxide (H2O2) also increased WDR35 expression in Neuro2a cells. Antioxidant (EUK-8) and p38 MAPK inhibitor (SB202190) treatment attenuated the increase in caspase-3 activity, cell death and WDR35 expression induced by bupivacaine or H2O2. Although transfection of Neuro2a cells with WDR35 siRNA attenuated the bupivacaine- or H2O2-induced increase in expression of WDR35 mRNA and protein, in contrast to our previous studies, it did not inhibit the increase in caspase-3 activity in bupivacaine- or H2O2-treated cells. Conclusions In summary, our results indicated that bupivacaine induced apoptosis in Neuro2a cells. Bupivacaine induced ROS generation and p38 MAPK activation, resulting in an increase in WDR35 expression, in these cells. However, the increase in WDR35 expression may not be essential for the bupivacaine-induced apoptosis in Neuro2a cells. These results may suggest the existence of another mechanism of bupivacaine-induced apoptosis independent from WDR35 expression in Neuro2a cells. PMID:23227925

Computational Systems Biology Approach Predicts Regulators and Targets of microRNAs and Their Genomic Hotspots in Apoptosis Process.

PubMed

Alanazi, Ibrahim O; Ebrahimie, Esmaeil

2016-07-01

Novel computational systems biology tools such as common targets analysis, common regulators analysis, pathway discovery, and transcriptomic-based hotspot discovery provide new opportunities in understanding of apoptosis molecular mechanisms. In this study, after measuring the global contribution of microRNAs in the course of apoptosis by Affymetrix platform, systems biology tools were utilized to obtain a comprehensive view on the role of microRNAs in apoptosis process. Network analysis and pathway discovery highlighted the crosstalk between transcription factors and microRNAs in apoptosis. Within the transcription factors, PRDM1 showed the highest upregulation during the course of apoptosis, with more than 9-fold expression increase compared to non-apoptotic condition. Within the microRNAs, MIR1208 showed the highest expression in non-apoptotic condition and downregulated by more than 6 fold during apoptosis. Common regulators algorithm showed that TNF receptor is the key upstream regulator with a high number of regulatory interactions with the differentially expressed microRNAs. BCL2 and AKT1 were the key downstream targets of differentially expressed microRNAs. Enrichment analysis of the genomic locations of differentially expressed microRNAs led us to the discovery of chromosome bands which were highly enriched (p < 0.01) with the apoptosis-related microRNAs, such as 13q31.3, 19p13.13, and Xq27.3 This study opens a new avenue in understanding regulatory mechanisms and downstream functions in the course of apoptosis as well as distinguishing genomic-enriched hotspots for apoptosis process.

Long non-coding RNA CASC15 promotes tongue squamous carcinoma progression through targeting miR-33a-5p.

PubMed

Zuo, Zhibin; Ma, Long; Gong, Zuode; Xue, Lande; Wang, Qibao

2018-05-26

Long non-coding RNAs (lncRNAs) have gained a lot of attention because they participate in several human disorders, including tumors. This study determined the role of LncRNA CASC15 (cancer susceptibility candidate 15) in the development of tongue squamous cell carcinoma (TSCC). Here, we identified that CASC15 expression was upregulated in TSCC samples and cell lines. We showed that overexpression of CASC15 promoted cell proliferation, cycle, and migration in TSCC. In addition, we revealed that miR-33a-5p expression was downregulated in TSCC tissues and cell lines. Moreover, we showed that the expression of CASC15 was negatively related with miR-33a-5p expression in TSCC tissues. Ectopic expression of miR-33a-5p suppressed cell proliferation, cycle, and migration in TSCC. Elevated expression of CASC15 suppressed miR-33a-5p expression and promoted ZEB1 expression in SCC4 cell. Ectopic expression of CASC15 promoted TSCC cell proliferation, cycle, and migration through targeting miR-33a-5p. These results suggested that lncRNA CASC15 and miR-33a-5p might be exploited as new markers of TSCC and were potential treatment targets for TSCC patients.

CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface fas ligand expression and amplifies fas-mediated hepatocyte death during allograft rejection.

PubMed

Afford, S C; Randhawa, S; Eliopoulos, A G; Hubscher, S G; Young, L S; Adams, D H

1999-01-18

We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68(+) macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.

Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasegawa, Kazuhiro; Wakino, Shu; Yoshioka, Kyoko

2008-07-18

NAD{sup +}-dependent protein deacetylase Sirt1 regulates cellular apoptosis. We examined the role of Sirt1 in renal tubular cell apoptosis by using HK-2 cells, proximal tubular cell lines with or without reactive oxygen species (ROS), H{sub 2}O{sub 2}. Without any ROS, Sirt1 inhibitors enhanced apoptosis and the expression of ROS scavenger, catalase, and Sirt1 overexpression downregulated catalase. When apoptosis was induced with H{sub 2}O{sub 2}, Sirt1 was upregulated with the concomitant increase in catalase expression. Sirt1 overexpression rescued H{sub 2}O{sub 2}-induced apoptosis through the upregulation of catalase. H{sub 2}O{sub 2} induced the nuclear accumulation of forkhead transcription factor, FoxO3a and themore » gene silencing of FoxO3a enhanced H{sub 2}O{sub 2}-induced apoptosis. In conclusion, endogenous Sirt1 maintains cell survival by regulating catalase expression and by preventing the depletion of ROS required for cell survival. In contrast, excess ROS upregulates Sirt1, which activates FoxO3a and catalase leading to rescuing apoptosis. Thus, Sirt1 constitutes a determinant of renal tubular cell apoptosis by regulating cellular ROS levels.« less

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease.

PubMed

Akie, Thomas E; Liu, Lijun; Nam, Minwoo; Lei, Shi; Cooper, Marcus P

2015-01-01

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance--ROS and PKCε activity--were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.

Elevated ATF4 Expression, in the Absence of Other Signals, Is Sufficient for Transcriptional Induction via CCAAT Enhancer-binding Protein-activating Transcription Factor Response Elements*

PubMed Central

Shan, Jixiu; Örd, Daima; Örd, Tõnis; Kilberg, Michael S.

2009-01-01

Protein limitation in vivo or amino acid deprivation of cells in culture causes a signal transduction cascade consisting of activation of the kinase GCN2 (general control nonderepressible 2), phosphorylation of eukaryotic initiation factor 2, and increased synthesis of activating transcription factor (ATF) 4 by a translational control mechanism. In a self-limiting transcriptional program, ATF4 transiently activates a wide range of downstream target genes involved in transport, cellular metabolism, and other cell functions. Simultaneous activation of other signal transduction pathways by amino acid deprivation led to the question of whether or not the increased abundance of ATF4 alone was sufficient to trigger the transcriptional control mechanisms. Using 293 cells that ectopically express ATF4 in a tetracycline-inducible manner showed that ATF4 target genes were activated in the absence of amino acid deprivation. Ectopic expression of ATF4 alone resulted in effective recruitment of the general transcription machinery, but some reduction in histone modification was observed. These data document that ATF4 alone is sufficient to trigger the amino acid-responsive transcriptional control program. However, the absolute amount of ectopic ATF4 required to achieve the same degree of transcriptional activation observed after amino acid limitation was greater, suggesting that other factors may serve to enhance ATF4 function. PMID:19509279

NF-κB is involved in the LPS-mediated proliferation and apoptosis of MAC-T epithelial cells as part of the subacute ruminal acidosis response in cows.

PubMed

Fan, Wen-Jie; Li, He-Ping; Zhu, He-Shui; Sui, Shi-Ping; Chen, Pei-Ge; Deng, Yue; Sui, Tong-Ming; Wang, Yue-Ying

2016-11-01

To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T. MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells. LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.

Transient development of ovotestes in XX Sox9 transgenic mice.

PubMed

Gregoire, Elodie P; Lavery, Rowena; Chassot, Anne-Amandine; Akiyama, Haruhiko; Treier, Mathias; Behringer, Richard R; Chaboissier, Marie-Christine

2011-01-01

The sex of an individual results from the paternal transmission of the SRY gene located on the Y chromosome. In turn, SRY initiates Sox9 expression, a transcription factor required for testicular differentiation. Ectopic activation of SOX9 in XX Wt1:Sox9 transgenic mice induces female-to-male sex reversal in adult mice. Here we show that complete sex reversal is preceded by a transient phase of ovotestis differentiation with XX Wt1:Sox9 transgenic gonads containing a testicular central region and one or both ovarian poles indicating that Wt1:Sox9 is not as efficient as Sry to induce male development. In XX Wt1:Sox9(Tg/+) gonads, transgenic Sox9 is expressed earlier than Sox9 in XY gonads and is able to induce the expression of EGFP, knocked into the 3' UTR of Sox9 indicating that SOX9 is involved in the initiation and maintenance of its own expression. However, the delayed onset of expression of endogenous Sox9-EGFP suggests that this activation requires other factors, whose expression depends on SOX9. In the testicular regions of the XX Wt1:Sox9 ovotestes, proliferation of the XX fetal germ cells is hampered and they differentiate as pro-spermatogonia. This indicates that XX germ cells are not competent to respond to proliferative signals released from a testicular environment. In the ovarian regions, despite the continuous mRNA expression of the WT1:Sox9 transgene, the SOX9 protein does not accumulate suggesting that regulation of this gene in ovarian cells involves post-transcriptional mechanisms. Finally, ovarian cells of the XX Wt1:Sox9 ovotestis undergo apoptosis during late embryogenesis leading to complete female-to-male sex reversal of the transgenic mice at birth. Copyright © 2010 Elsevier Inc. All rights reserved.

miR-330 regulates the proliferation of colorectal cancer cells by targeting Cdc42

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yuefeng; Zhu, Xiaolan; Xu, Wenlin

2013-02-15

Highlights: ► miR-330 was inversely correlated with Cdc42 in colorectal cancer cells. ► Elevated miR-330 suppressed cell proliferation in vivo and in vitro. ► Elevated miR-330 mimicked the effect of Cdc42 knockdown. ► Restoration of Cdc42 could partially attenuate the effects of miR-330. -- Abstract: MicroRNAs are small non-coding RNA molecules that play important roles in the multistep process of colorectal carcinoma (CRC) development. However, the miRNA–mRNA regulatory network is far from being fully understood. The objective of this study was to investigate the expression and the biological roles of miR-330 in colorectal cancer cells. Cdc42, one of the bestmore » characterized members of the Rho GTPase family, was found to be up-regulated in several types of human tumors including CRC and has been implicated in cancer initiation and progression. In the present study, we identified miR-330, as a potential regulator of Cdc42, was found to be inversely correlated with Cdc42 expression in colorectal cancer cell lines. Ectopic expression of miR-330 down-regulated Cdc42 expression at both protein and mRNA level, mimicked the effect of Cdc42 knockdown in inhibiting proliferation, inducing G1 cell cycle arrest and apoptosis of the colorectal cancer cells, whereas restoration of Cdc42 could partially attenuate the effects of miR-330. In addition, elevated expression of miR-330 could suppress the immediate downstream effectors of Cdc42 and inhibit the growth of colorectal cancer cells in vivo. To sum up, our results establish a role of miR-330 in negatively regulating Cdc42 expression and colorectal cancer cell proliferation. They suggest that manipulating the expression level of Cdc42 by miR-330 has the potential to influence colorectal cancer progression.« less

LRIG1, a 3p tumor suppressor, represses EGFR signaling and is a novel epigenetic silenced gene in colorectal cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kou, Changhua, E-mail: chkoukou@hotmail.com; Zhou, Tian; Han, Xilin

2015-08-21

Downregulation of LRIG1 was found in many types of cancer. However, data concerning the possible mechanism of LRIG1 reduction in cancers were not reported yet. To analyze the regulation and function of LRIG1 in colorectal cancer (CRC), 6 cell lines, 46 paired tissues from primary CRC cases were employed in this study. In CRC cell lines, under-expression of LRIG1 was correlated with promoter region hypermethylation, and restoration of LRIG1 was induced by 5-Aza-2'-deoxyazacytidine treatment. Subsequently, we ectopically expressed LRIG1 in LRIG1 low-expressing HCT-116 cells and suppressed LRIG1 in LRIG1 high-expressing LoVo cells. We found that over-expression of LRIG1 inhibits cellmore » proliferation and colony formation and tumor growth, while knockdown of LRIG1 promotes cell proliferation and colony formation. Decreased and increased EGFR/AKT signaling pathway may partially explain the lower and higher rates of proliferation in CRC cells transfected with LRIG1 cDNA or shRNA. In clinical samples, we compared the methylation, mRNA and protein expression of LRIG1 in samples of CRC tissues. A significant increase in LRIG1 methylation was identified in CRC specimens compared to adjacent normal tissues and that it was negatively correlated with its mRNA and protein expression. In conclusion, LRIG1 is frequently methylated in human CRC and consequent mRNA and protein downregulation may contribute to tumor growth by activating EGFR/AKT signaling. - Highlights: • Promoter methylation of LRIG1 occurred in colorectal cancer cells and tumors. • Restoration of LRIG1 inhibits tumor growth in vitro and in vivo. • Overexpression or knockdown of LRIG1 regulates EGFR/AKT and downstream apoptosis. • Methylation of LRIG1 correlates with its mRNA and protein downregulation. • LRIG1 was firstly identified as an epigenetic target in cancer.« less

Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

PubMed

Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

2015-12-01

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. Copyright © 2015 by the Genetics Society of America.

Auranofin-mediated inhibition of PI3K/AKT/mTOR axis and anticancer activity in non-small cell lung cancer cells

PubMed Central

Li, Hongyu; Hu, Jing; Wu, Shuhong; Wang, Li; Cao, Xiaobo; Zhang, Xiaoshan; Dai, Bingbing; Cao, Mengru; Shao, Ruping; Zhang, Ran; Majidi, Mourad; Ji, Lin; Heymach, John V.; Wang, Michael; Pan, Shiyang; Minna, John; Mehran, Reza J.; Swisher, Stephen G.; Roth, Jack A.; Fang, Bingliang

2016-01-01

Auranofin, a gold complex that has been used to treat rheumatoid arthritis in clinics and has documented pharmacokinetic and safety profiles in humans, has recently been investigated for its anticancer activity in leukemia and some solid cancers. However, auranofin's single agent activity in lung cancer is not well characterized. To determine whether auranofin has single agent activity in lung cancer, we evaluated auranofin's activity in a panel of 10 non-small cell lung cancer (NSCLC) cell lines. Cell viability analysis revealed that auranofin induced growth inhibition in a subset of NSCLC cell lines with a half maximal inhibitory concentration (IC50) below 1.0 μM. Treatment with auranofin elicited apoptosis and necroptosis in auranofin-sensitive cell lines. Moreover, the susceptibility of NSCLC cells to auranofin was inversely correlated with TXNRD1 expression in the cells. Transient transfection of the TXNRD1-expressing plasmid in auranofin-sensitive Calu3 cells resulted in partial resistance, indicating that high TXNRD level is one of causal factors for resistance to auranofin. Further mechanistic characterization with proteomic analysis revealed that auranofin inhibits expression and/or phosphorylation of multiple key nodes in the PI3K/AKT/mTOR pathway, including S6, 4EBP1, Rictor, p70S6K, mTOR, TSC2, AKT and GSK3. Ectopic expression of TXNRD1 partially reversed auranofin-mediated PI3K/AKT/mTOR inhibition, suggesting that TXNRD1 may participate in the regulation of PI3K/AKT/mTOR pathway. Administration of auranofin to mice with xenograft tumors derived from NSCLC cells significantly suppressed tumor growth without inducing obvious toxic effects. Our results demonstrated feasibility of repurposing auranofin for treatment of lung cancer. PMID:26657290

Intrafibrillar-silicified collagen scaffolds enhance the osteogenic capacity of human dental pulp stem cells.

PubMed

Niu, Li-na; Sun, Jia-qi; Li, Qi-hong; Jiao, Kai; Shen, Li-juan; Wu, Dan; Tay, Franklin; Chen, Ji-hua

2014-07-01

The present study investigated the effects of intrafibrillar-silicified collagen scaffolds (ISCS) on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo. The hDPSCs were co-cultured with ISCS or nonsilicified collagen scaffolds (NCS) in control medium (CM) or osteogenic differentiation medium (ODM). Cell cycle and cell apoptosis were analyzed with flow cytometry to measure the viability of hDPSCs. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to evaluate the expression levels of osteogenic marker genes and proteins of hDPSCs. Alkaline phosphatase (ALP) staining and alizarin red S assay were used to evaluate the ALP activity of hDPSCs and their calcium deposition potential. In addition, hDPSCs and scaffolds were implanted subcutaneously in nude mice for 8 weeks. Harvested tissues were immunohistochemically stained for osteocalcin (OCN) expression from hDPSCs, and stained with alizarin red S for examination of their calcium deposition in vivo. The ISCS had no adverse effect on hDPSCs, promoted their proliferation, and significantly up-regulated the expression of osteogenesis-related genes and proteins. The hDPSCs co-cultured with ISCS in ODM exhibited the highest ALP activity and calcium deposition in vitro. The ISCS promoted the OCN expression and calcium deposition of hDPSCs after ectopic transplantation in vivo. Intrafibrillar-silicified collagen scaffolds significantly promoted the proliferation, osteogenic differentiation and mineralization of hDPSCs, when compared with NCS. This study demonstrates combining the use of hDPSCs and ISCS to promote bone-like tissue formation is a promising approach for clinical bone repair and regeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Novel Post-translational Modification of Nucleolin, SUMOylation at Lys-294, Mediates Arsenite-induced Cell Death by Regulating gadd45α mRNA Stability*

PubMed Central

Zhang, Dongyun; Liang, Yuguang; Xie, Qipeng; Gao, Guangxun; Wei, Jinlong; Huang, Haishan; Li, Jingxia; Gao, Jimin; Huang, Chuanshu

2015-01-01

Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation. PMID:25561743

A novel mechanism by which tissue transglutaminase activates signaling events that promote cell survival.

PubMed

Boroughs, Lindsey K; Antonyak, Marc A; Cerione, Richard A

2014-04-04

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Targeting MYCN-Driven Transcription By BET-Bromodomain Inhibition.

PubMed

Henssen, Anton; Althoff, Kristina; Odersky, Andrea; Beckers, Anneleen; Koche, Richard; Speleman, Frank; Schäfers, Simon; Bell, Emma; Nortmeyer, Maike; Westermann, Frank; De Preter, Katleen; Florin, Alexandra; Heukamp, Lukas; Spruessel, Annika; Astrahanseff, Kathy; Lindner, Sven; Sadowski, Natalie; Schramm, Alexander; Astorgues-Xerri, Lucile; Riveiro, Maria E; Eggert, Angelika; Cvitkovic, Esteban; Schulte, Johannes H

2016-05-15

Targeting BET proteins was previously shown to have specific antitumoral efficacy against MYCN-amplified neuroblastoma. We here assess the therapeutic efficacy of the BET inhibitor, OTX015, in preclinical neuroblastoma models and extend the knowledge on the role of BRD4 in MYCN-driven neuroblastoma. The efficacy of OTX015 was assessed in in vitro and in vivo models of human and murine MYCN-driven neuroblastoma. To study the effects of BET inhibition in the context of high MYCN levels, MYCN was ectopically expressed in human and murine cells. The effect of OTX015 on BRD4-regulated transcriptional pause release was analyzed using BRD4 and H3K27Ac chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and gene expression analysis in neuroblastoma cells treated with OTX015 compared with vehicle control. OTX015 showed therapeutic efficacy against preclinical MYCN-driven neuroblastoma models. Similar to previously described BET inhibitors, concurrent MYCN repression was observed in OTX015-treated samples. Ectopic MYCN expression, however, did not abrogate effects of OTX015, indicating that MYCN repression is not the only target of BET proteins in neuroblastoma. When MYCN was ectopically expressed, BET inhibition still disrupted MYCN target gene transcription without affecting MYCN expression. We found that BRD4 binds to super-enhancers and MYCN target genes, and that OTX015 specifically disrupts BRD4 binding and transcription of these genes. We show that OTX015 is effective against mouse and human MYCN-driven tumor models and that BRD4 not only targets MYCN, but specifically occupies MYCN target gene enhancers as well as other genes associated with super-enhancers. Clin Cancer Res; 22(10); 2470-81. ©2015 AACR. ©2015 American Association for Cancer Research.

Ectopic expression of UGT84A2 delayed flowering by indole-3-butyric acid-mediated transcriptional repression of ARF6 and ARF8 genes in Arabidopsis.

PubMed

Zhang, Gui-Zhi; Jin, Shang-Hui; Li, Pan; Jiang, Xiao-Yi; Li, Yan-Jie; Hou, Bing-Kai

2017-12-01

Ectopic expression of auxin glycosyltransferase UGT84A2 in Arabidopsis can delay flowering through increased indole-3-butyric acid and suppressed transcription of ARF6, ARF8 and flowering-related genes FT, SOC1, AP1 and LFY. Auxins are critical regulators for plant growth and developmental processes. Auxin homeostasis is thus an important issue for plant biology. Here, we identified an indole-3-butyric acid (IBA)-specific glycosyltransferase, UGT84A2, and characterized its role in Arabidopsis flowering development. UGT84A2 could catalyze the glycosylation of IBA, but not indole-3-acetic acid (IAA). UGT84A2 transcription expression was clearly induced by IBA. When ectopically expressing in Arabidopsis, UGT84A2 caused obvious delay in flowering. Correspondingly, the increase of IBA level, the down-regulation of AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8, and the down-regulation of flowering-related genes such as FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO1(SOC1), APETALA1 (AP1), and LEAFY(LFY) were observed in transgenic plants. When exogenously applying IBA to wild-type plants, the late flowering phenotype, the down-regulation of ARF6, ARF8 and flowering-related genes recurred. We examined the arf6arf8 double mutants and found that the expression of flowering-related genes was also substantially decreased in these mutants. Together, our results suggest that glycosyltransferase UGT84A2 may be involved in flowering regulation through indole-3-butyric acid-mediated transcriptional repression of ARF6, ARF8 and downstream flowering pathway genes.

Glycogen synthase kinase 3 regulates PAX3-FKHR-mediated cell proliferation in human alveolar rhabdomyosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Fu-Yue; Dong, Hanqing; Cui, Jimmy

2010-01-01

Patients with alveolar rhabdomyosarcoma (ARMS) have poorer response to conventional chemotherapy and lower survival rates than those with embryonal RMS (ERMS). To identify compounds that preferentially block the growth of ARMS, we conducted a small-scale screen of 160 kinase inhibitors against the ARMS cell line Rh30 and ERMS cell line RD and identified inhibitors of glycogen synthase kinase 3 (GSK3), including TWS119 as ARMS-selective inhibitors. GSK3 inhibitors inhibited cell proliferation and induced apoptosis more effectively in Rh30 than RD cells. Ectopic expression of fusion protein PAX3-FKHR in RD cells significantly increased their sensitivity to TWS119. Down-regulation of GSK3 by GSK3more » inhibitors or siRNA significantly reduced the transcriptional activity of PAX3-FKHR. These results suggest that GSK3 is directly involved in regulating the transcriptional activity of PAX3-FKHR. Also, GSK3 phosphorylated PAX3-FKHR in vitro, suggesting that GSK3 might regulate PAX3-FKHR activity via phosphorylation. These findings support a novel mechanism of PAX3-FKHR regulation by GSK3 and provide a novel strategy to develop GSK inhibitors as anti-ARMS therapies.« less

Drosophila bunched integrates opposing DPP and EGF signals to set the operculum boundary.

PubMed

Dobens, L L; Peterson, J S; Treisman, J; Raftery, L A

2000-02-01

The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Here we show that opposing DPP and EGF signals set the boundary for an ovarian follicle cell fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein MAD; ectopic expression of BUNCHED suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. We previously demonstrated that the bunched expression pattern requires the EGF receptor ligand GURKEN. Here we show that activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. We propose that the juxtaposition of cells with high and low BUNCHED activity organizes a sharp boundary for the operculum fate.

Cold Inducible RNA Binding Protein Is Involved in Chronic Hypoxia Induced Neuron Apoptosis by Down-Regulating HIF-1α Expression and Regulated By microRNA-23a.

PubMed

Chen, Xiaoming; Liu, Xinqin; Li, Bin; Zhang, Qian; Wang, Jiye; Zhang, Wenbin; Luo, Wenjing; Chen, Jingyuan

2017-01-01

Background: Neuron apoptosis mediated by hypoxia inducible factor 1α (HIF-1α) in hippocampus is one of the most important factors accounting for the chronic hypobaric hypoxia induced cognitive impairment. As a neuroprotective molecule that is up-regulated in response to various environmental stress, CIRBP was reported to crosstalk with HIF-1α under cellular stress. However, its function under chronic hypobaric hypoxia remains unknown. Objective: In this study, we tried to identify the role of CIRBP in HIF-1α mediated neuron apoptosis under chronic hypobaric hypoxia and find a possible method to maintain its potential neuroprotective in long-term high altitude environmental exposure. Methods: We established a chronic hypobaric hypoxia rat model as well as a tissue culture model where SH-SY5Y cells were exposed to 1% hypoxia. Based on these models, we measured the expressions of HIF-1α and CIRBP under hypoxia exposure and examined the apoptosis of neurons by TUNEL immunofluorescence staining and western blot analysis of apoptosis related proteins. In addition, by establishing HIF-1α shRNA and pEGFP-CIRBP plasmid transfected cells, we confirmed the role of HIF-1α in chronic hypoxia induced neuron apoptosis and identified the influence of CIRBP over-expression upon HIF-1α and neuron apoptosis in the process of exposure. Furthermore, we measured the expression of the reported hypoxia related miRNAs in both models and the influence of miRNAs' over-expression/knock-down upon CIRBP in the process of HIF-1α mediated neuron apoptosis. Results: HIF-1α expression as well as neuron apoptosis was significantly elevated by chronic hypobaric hypoxia both in vivo and in vitro . CIRBP was induced in the early stage of exposure (3d/7d); however as the exposure was prolonged (21d), CIRBP level of the hypoxia group became significantly lower than that of control. In addition, HIF-1α knockdown significantly decreased neuron apoptosis under hypoxia, suggesting HIF-1α may be pro-apoptotic in the process of exposure. CIRBP over-expression significantly suppressed HIF-1α up-regulation in hypoxia and inhibited HIF-1α mediated neuron apoptosis. Interestingly, miR-23a was also induced by hypoxia exposure and showed the same changing tendency with CIRBP (increasing in 3d/7d, decreasing in 21d). In addition, over-expressing miR-23a up-regulated CIRBP, down-regulated HIF-1α and attenuated neuron apoptosis. Conclusion: Cold inducible RNA binding protein is involved in chronic hypoxia induced neuron apoptosis by down-regulating HIF-1α expression, and MiR-23a may be an important tool to maintain CIRBP level and function.

Autoimmune Regulator (AIRE) Is Expressed in Spermatogenic Cells, and It Altered the Expression of Several Nucleic-Acid-Binding and Cytoskeletal Proteins in Germ Cell 1 Spermatogonial (GC1-spg) Cells.

PubMed

Radhakrishnan, Karthika; Bhagya, Kongattu P; Kumar, Anil Tr; Devi, Anandavalli N; Sengottaiyan, Jeeva; Kumar, Pradeep G

2016-08-01

Autoimmune regulator (AIRE) is a gene associated with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE is expressed heavily in the thymic epithelial cells and is involved in maintaining self-tolerance through regulating the expression of tissue-specific antigens. The testes are the most predominant extrathymic location where a heavy expression of AIRE is reported. Homozygous Aire-deficient male mice were infertile, possibly due to impaired spermatogenesis, deregulated germ cell apoptosis, or autoimmunity. We report that AIRE is expressed in the testes of neonatal, adolescent, and adult mice. AIRE expression was detected in glial cell derived neurotrophic factor receptor alpha (GFRα)(+) (spermatogonia), GFRα(-)/synaptonemal complex protein (SCP3)(+) (meiotic), and GFRα(-)/Phosphoglycerate kinase 2 (PGK2)(+) (postmeiotic) germ cells in mouse testes. GC1-spg, a germ-cell-derived cell line, did not express AIRE. Retinoic acid induced AIRE expression in GC1-spg cells. Ectopic expression of AIRE in GC1-spg cells using label-free LC-MS/MS identified a total of 371 proteins that were differentially expressed. 100 proteins were up-regulated, and 271 proteins were down-regulated. Data are available via ProteomeXchange with identifier PXD002511. Functional analysis of the differentially expressed proteins showed increased levels of various nucleic-acid-binding proteins and transcription factors and a decreased level of various cytoskeletal and structural proteins in the AIRE overexpressing cells as compared with the empty vector-transfected controls. The transcripts of a select set of the up-regulated proteins were also elevated. However, there was no corresponding decrease in the mRNA levels of the down-regulated set of proteins. Molecular function network analysis indicated that AIRE influenced gene expression in GC1-spg cells by acting at multiple levels, including transcription, translation, RNA processing, protein transport, protein localization, and protein degradation, thus setting the foundation in understanding the functional role of AIRE in germ cell biology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Endometrial seedlings. A survival instinct? Immunomodulation and its role in the pathophysiology of endometriosis.

PubMed

Portelli, M; Pollacco, J; Sacco, K; Schembri-Wismayer, P; Calleja-Agius, J

2011-12-01

Endometriosis occurs when ectopic cells from the endometrium implant within the peritoneum. It is considered as a disease of multifactorial aetiology and affects 7-10% of women of reproductive age worldwide. In endometriosis, the immune system is thought to be dysfunctional and various studies have shown cytokine imbalance. Commonly upregulated cytokines include Tumour necrosis factor-alpha, interferon gamma and interleukin-10. Through analysis of the molecular makeup of the peritoneal fluid, a change is shown to occur, conferring resistance from macrophages and lymphocytes to endometrial cells. This is possibly due to a reduced Inter-cellular adhesion molecule-1 synthesis. Survival of ectopic endometrial cells also arises through the expression of human leukocyte antigens. Apart from the survival of ectopic/eutopic cells in endometriosis, there is marked cellular proliferation, which has also been attributed to a change in the expression of proteins such as Bcl-2-Associated X protein, B-cell lymphoma-2 protein, transforming growth factor-beta and the enzyme aromatase. Danazol and aromatase inhibitors modulate the immune system, thus allowing partial restoration of cytokine levels. Pharmacogenomics may be the way forward in developing novel treatment modalities for endometriosis.

Nerve-independent and ectopically additional induction of taste buds in organ culture of fetal tongues.

PubMed

Honda, Kotaro; Tomooka, Yasuhiro

2016-10-01

An improved organ culture system allowed to observe morphogenesis of mouse lingual papillae and taste buds relatively for longer period, in which fetal tongues were analyzed for 6 d. Taste cells were defined as eosinophobic epithelial cells expressing CK8 and Sox2 within lingual epithelium. Addition of glycogen synthase kinase 3 beta inhibitor CHIR99021 induced many taste cells and buds in non-gustatory and gustatory stratified lingual epithelium. The present study clearly demonstrated induction of taste cells and buds ectopically and without innervation.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector.

PubMed

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis -specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

PubMed Central

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria. PMID:28638803

The tripartite leader sequence is required for ectopic expression of HAdV-B and HAdV-E E3 CR1 genes.

PubMed

Bair, Camden R; Kotha Lakshmi Narayan, Poornima; Kajon, Adriana E

2017-05-01

The unique repertoire of genes that characterizes the early region 3 (E3) of the different species of human adenovirus (HAdV) likely contributes to their distinct pathogenic traits. The function of many E3 CR1 proteins remains unknown possibly due to unidentified intrinsic properties that make them difficult to express ectopically. This study shows that the species HAdV-B- and HAdV-E-specific E3 CR1 genes can be expressed from vectors carrying the HAdV tripartite leader (TPL) sequence but not from traditional mammalian expression vectors. Insertion of the TPL sequence upstream of the HAdV-B and HAdV-E E3 CR1 open reading frames was sufficient to rescue protein expression from pCI-neo constructs in transfected 293T cells. The detection of higher levels of HAdV-B and HAdV-E E3 CR1 transcripts suggests that the TPL sequence may enhance gene expression at both the transcriptional and translational levels. Our findings will facilitate the characterization of additional AdV E3 proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

hnRNP K plays a protective role in TNF-α-induced apoptosis in podocytes.

PubMed

Zhao, Shili; Feng, Junxia; Wang, Qi; Tian, Lu; Zhang, Yunfang; Li, Hongyan

2018-06-29

Apoptosis of podocytes contributes to proteinuria in many chronic kidney diseases. The cytokine, tumor necrosis factor-α (TNF-α) is thought to be involved in podocyte apoptosis, but the underlying mechanism is not understood. In our study, we established a model of TNF-α-induced apoptosis by isolating primary podocytes from mice. After exposing cells to TNF-α, we determined the expression levels of heterogeneous nuclear ribonucleoprotein K (hnRNP K) and cellular FLICE-inhibitory protein (c-FLIP) and the phosphorylation levels of glycogen synthase kinase β (GSK3β) and extracellular signal-regulated kinase (ERK). We then knocked down or overexpressed the levels of hnRNP K and observed its effects on the expressions of c-FLIP, caspase-8, caspase-3, and the phosphorylation of GSK3β and ERK. In addition, we examined the percentage of cells undergoing apoptosis and studied cell cycle distribution. We found that TNF-α induced apoptosis in podocytes and that the expressions of hnRNP K and c-FLIP were significantly decreased, whereas the phosphorylations of GSK3β and ERK were significantly increased. Both gene knockdown and overexpression of hnRPN K resulted in varied expressions/phosphorylations of c-FLIP, GSK3β, and ERK. Moreover, decreased hnRPN K expression contributed to increased levels of caspase-8 and capase-3, as well as an increase in cell apoptosis and G0/G1 arrest. In conclusion, down-regulated expression of hnRNP K by TNF-α resulted in a decrease in the expression of c-FLIP as well as increases in phosphorylated GSK3β, ERK, caspase-8, and caspase-3, and then critically contributed to the podocyte apoptosis. © 2018 The Author(s).

Tributyltin increases the expression of apoptosis- and adipogenesis-related genes in rat ovaries

PubMed Central

Lee, Hyojin; Lim, Sojeong; Yun, Sujin; Yoon, Ayoung; Park, Gayoung

2012-01-01

Objective Tributyltin (TBT), an endocrine disrupting chemical, has been reported to decrease ovarian function by causing apoptosis in the ovary, but the mechanism is not fully understood. Therefore, we examined whether TBT increases the expression of adipogenesis-related genes in the ovary and the increased expression of these genes is associated with apoptosis induction. Methods Three-week-old Sprague-Dawley rats were orally administered TBT (1 or 10 mg/kg body weight) or sesame oil as a control for 7 days. The ovaries were obtained and weighed on day 8, and then they were fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or frozen for RNA extraction. Using the total RNA of the ovaries, adipogenesis- and apoptosis-related genes were analyzed by real-time polymerase chain reaction (PCR). Results The ovarian weight was significantly decreased in rats administered 10 mg/kg TBT compared to that in control rats. As determined by the TUNEL assay, the number of apoptotic follicles in ovary was significantly increased in rats administered 10 mg/kg TBT. The real-time PCR results showed that the expression of adipogenesis-related genes such as PPARγ, aP2, CD36, and PEPCK was increased after TBT administration. In addition, apoptosis-related genes such as TNFα and TNFR1 were expressed more in the TBT-administered rats compared with the control rats. Conclusion The present study demonstrates that TBT induces the expression of adipogenesis- and apoptosis-related genes in the ovary leading to apoptosis in the ovarian follicles. These results suggest that the increased expression of adipogenesis-related genes in the ovary by TBT exposure might induce apoptosis resulting in a loss of ovarian function. PMID:22563546

The myogenic repressor gene Holes in muscles is a direct transcriptional target of Twist and Tinman in the Drosophila embryonic mesoderm

PubMed Central

Elwell, Jennifer A.; Lovato, TyAnna L.; Adams, Melanie M.; Baca, Erica M.; Lee, Thai; Cripps, Richard M.

2015-01-01

Understanding the regulatory circuitry controlling myogenesis is critical to understanding developmental mechanisms and developmentally-derived diseases. We analyzed the transcriptional regulation of a Drosophila myogenic repressor gene, Holes in muscles (Him). Previously, Him was shown to inhibit Myocyte enhancer factor-2 (MEF2) activity, and is expressed in myoblasts but not differentiating myotubes. We demonstrate that different phases of Him embryonic expression arise through the actions of different enhancers, and we characterize the enhancer required for its early mesoderm expression. This Him early mesoderm enhancer contains two conserved binding sites for the basic helix-loop-helix regulator Twist, and one binding site for the NK homeodomain protein Tinman. The sites for both proteins are required for enhancer activity in early embryos. Twist and Tinman activate the enhancer in tissue culture assays, and ectopic expression of either factor is sufficient to direct ectopic expression of a Him-lacZ reporter, or of the endogenous Him gene. Moreover, sustained expression of twist expression in the mesoderm up-regulates mesodermal Him expression in late embryos. Our findings provide a model to define mechanistically how Twist can both promotes myogenesis through direct activation of Mef2, and can place a brake on myogenesis, through direct activation of Him. PMID:25704510

Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells.

PubMed

Han, Min Ae; Min, Kyoung-Jin; Woo, Seon Min; Seo, Bo Ram; Kwon, Taeg Kyu

2016-10-04

Eupafolin, a flavone found in Artemisia princeps, has been reported for its anti-tumor activity in several cancer cells. In this study, we examined whether eupafolin could sensitize TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that eupafolin alone and TRAIL alone had no effect on apoptosis. However, combined treatment with eupafolin and TRAIL markedly induced apoptosis in human renal carcinoma (Caki) cells, glioma cells (U251MG), and prostate cancer cells (DU145), but not normal cells [mesangial cells (MC) and normal mouse kidney cells (TCMK-1)]. Eupafolin induced down-regulation of Mcl-1 expression at the post-translational levels in cathepsin S-dependent manner, and over-expression of Mcl-1 markedly blocked apoptosis induced by combined treatment with eupafolin and TRAIL. In addition, eupafolin increased Bim expression at the post-translational levels via AMP-activated protein kinase (AMPK)-mediated inhibition of proteasome activity. Knock-down of Bim expression by siRNA inhibited eupafolin plus TRAIL-induced apoptosis. Furthermore, combined treatment with eupafolin and TRAIL reduced tumor growth in xenograft models. Taken together, these results suggest that eupafolin enhanced TRAIL-mediated apoptosis via down-regulation of Mcl-1 and up-regulation of Bim in renal carcinoma Caki cells.

Eupafolin enhances TRAIL-mediated apoptosis through cathepsin S-induced down-regulation of Mcl-1 expression and AMPK-mediated Bim up-regulation in renal carcinoma Caki cells

PubMed Central

Woo, Seon Min; Seo, Bo Ram; Kwon, Taeg Kyu

2016-01-01

Eupafolin, a flavone found in Artemisia princeps, has been reported for its anti-tumor activity in several cancer cells. In this study, we examined whether eupafolin could sensitize TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that eupafolin alone and TRAIL alone had no effect on apoptosis. However, combined treatment with eupafolin and TRAIL markedly induced apoptosis in human renal carcinoma (Caki) cells, glioma cells (U251MG), and prostate cancer cells (DU145), but not normal cells [mesangial cells (MC) and normal mouse kidney cells (TCMK-1)]. Eupafolin induced down-regulation of Mcl-1 expression at the post-translational levels in cathepsin S-dependent manner, and over-expression of Mcl-1 markedly blocked apoptosis induced by combined treatment with eupafolin and TRAIL. In addition, eupafolin increased Bim expression at the post-translational levels via AMP-activated protein kinase (AMPK)-mediated inhibition of proteasome activity. Knock-down of Bim expression by siRNA inhibited eupafolin plus TRAIL-induced apoptosis. Furthermore, combined treatment with eupafolin and TRAIL reduced tumor growth in xenograft models. Taken together, these results suggest that eupafolin enhanced TRAIL-mediated apoptosis via down-regulation of Mcl-1 and up-regulation of Bim in renal carcinoma Caki cells. PMID:27582546

Decoy receptor 3 promotes cell adhesion and enhances endometriosis development.

PubMed

Tsai, Hsiao-Wen; Huang, Ming-Ting; Wang, Peng-Hui; Huang, Ben-Shian; Chen, Yi-Jen; Hsieh, Shie-Liang

2018-02-01

Endometriosis is a multifactorial inflammatory disease with persistent activation of the nuclear factor-κB (NF-κB) signalling pathway. Aberrant adhesion of endometrium is the essential step in the progression of endometriosis, but the molecular mechanism of ectopic growth of endometrium is still unclear. Decoy receptor 3 (DcR3)/TNFRSF6B, a pleiotropic immunomodulator regulated by oestrogen, is able to activate focal adhesion kinase to promote cell adhesion. We found that DcR3 is upregulated in human ectopic endometrial cells via activation of the Akt-NF-κB signalling pathway, and its expression level correlates positively with that of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and homing cell adhesion molecule (HCAM; CD44). In a multivariate regression model, DcR3 expression level was the most significant parameter associated with endometriosis severity. Knockdown of DcR3 not only downregulated the expression of ICAM-1 and HCAM, but also reduced cell adhesion and migration. In vivo investigation further showed that DcR3 promoted the growth and spread of endometrium, whereas knockdown of DcR3 by lentivirus-delivered short hairpin RNA inhibited ectopic adhesion of endometrium and abrogated endometriosis progression. These observations are in support of DcR3 playing a critical role in the pathogenesis of endometriosis, and the inhibition of DcR3 expression being a promising approach for the treatment of endometriosis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nonstructural proteins of respiratory syncytial virus suppress premature apoptosis by an NF-kappaB-dependent, interferon-independent mechanism and facilitate virus growth.

PubMed

Bitko, Vira; Shulyayeva, Olena; Mazumder, Barsanjit; Musiyenko, Alla; Ramaswamy, Murali; Look, Dwight C; Barik, Sailen

2007-02-01

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-kappaB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway.

Nonstructural Proteins of Respiratory Syncytial Virus Suppress Premature Apoptosis by an NF-κB-Dependent, Interferon-Independent Mechanism and Facilitate Virus Growth▿

PubMed Central

Bitko, Vira; Shulyayeva, Olena; Mazumder, Barsanjit; Musiyenko, Alla; Ramaswamy, Murali; Look, Dwight C.; Barik, Sailen

2007-01-01

The two nonstructural (NS) proteins NS1 and NS2 of respiratory syncytial virus (RSV) are abundantly expressed in the infected cell but are not packaged in mature progeny virions. We found that both proteins were expressed early in infection, whereas the infected cells underwent apoptosis much later. Coincident with NS protein expression, a number of cellular antiapoptotic factors were expressed or activated at early stages, which included NF-κB and phosphorylated forms of protein kinases AKT, phosphoinositide-dependent protein kinase, and glycogen synthase kinase. Using specific short interfering RNAs (siRNAs), we achieved significant knockdown of one or both NS proteins in the infected cell, which resulted in abrogation of the antiapoptotic functions and led to early apoptosis. NS-dependent suppression of apoptosis was observed in Vero cells that are naturally devoid of type I interferons (IFN). The siRNA-based results were confirmed by the use of NS-deleted RSV mutants. Early activation of epidermal growth factor receptor (EGFR) in the RSV-infected cell did not require NS proteins. Premature apoptosis triggered by the loss of NS or by apoptosis-promoting drugs caused a severe reduction of RSV growth. Finally, recombinantly expressed NS1 and NS2, individually and together, reduced apoptosis by tumor necrosis factor alpha, suggesting an intrinsic antiapoptotic property of both. We conclude that the early-expressed nonstructural proteins of RSV boost viral replication by delaying the apoptosis of the infected cell via a novel IFN- and EGFR-independent pathway. PMID:17151097

Metadherin facilitates podocyte apoptosis in diabetic nephropathy

PubMed Central

Liu, Wen-Ting; Peng, Fen-Fen; Li, Hong-Yu; Chen, Xiao-Wen; Gong, Wang-Qiu; Chen, Wen-Jing; Chen, Yi-Hua; Li, Pei-Lin; Li, Shu-Ting; Xu, Zhao-Zhong; Long, Hai-Bo

2016-01-01

Apoptosis, one of the major causes of podocyte loss, has been reported to have a vital role in diabetic nephropathy (DN) pathogenesis, and understanding the mechanisms underlying the regulation of podocyte apoptosis is crucial. Metadherin (MTDH) is an important oncogene, which is overexpressed in most cancers and responsible for apoptosis, metastasis, and poor patient survival. Here we show that the expression levels of Mtdh and phosphorylated p38 mitogen-activated protein kinase (MAPK) are significantly increased, whereas those of the microRNA-30 family members (miR-30s) are considerably reduced in the glomeruli of DN rat model and in high glucose (HG)-induced conditionally immortalized mouse podocytes (MPC5). These levels are positively correlated with podocyte apoptosis rate. The inhibition of Mtdh expression, using small interfering RNA, but not Mtdh overexpression, was shown to inhibit HG-induced MPC5 apoptosis and p38 MAPK pathway, and Bax and cleaved caspase 3 expression. This was shown to be similar to the effects of p38 MAPK inhibitor (SB203580). Furthermore, luciferase assay results demonstrated that Mtdh represents the target of miR-30s. Transient transfection experiments, using miR-30 microRNA (miRNA) inhibitors, led to the increase in Mtdh expression and induced the apoptosis of MPC5, whereas the treatment with miR-30 miRNA mimics led to the reduction in Mtdh expression and apoptosis of HG-induced MPC5 cells in comparison with their respective controls. Our results demonstrate that Mtdh is a potent modulator of podocyte apoptosis, and that it represents the target of miR-30 miRNAs, facilitating podocyte apoptosis through the activation of HG-induced p38 MAPK-dependent pathway. PMID:27882943

The role of ARK in stress-induced apoptosis in Drosophila cells

PubMed Central

Zimmermann, Katja C.; Ricci, Jean-Ehrland; Droin, Nathalie M.; Green, Douglas R.

2002-01-01

The molecular mechanisms of apoptosis are highly conserved throughout evolution. The homologs of genes essential for apoptosis in Caenorhabditis elegans and Drosophila melanogaster have been shown to be important for apoptosis in mammalian systems. Although a homologue for CED-4/apoptotic protease-activating factor (Apaf)-1 has been described in Drosophila, its exact function and the role of the mitochondrial pathway in its activation remain unclear. Here, we used the technique of RNA interference to dissect apoptotic signaling pathways in Drosophila cells. Inhibition of the Drosophila CED-4/Apaf-1–related killer (ARK) homologue resulted in pronounced inhibition of stress-induced apoptosis, whereas loss of ARK did not protect the cells from Reaper- or Grim-induced cell death. Reduction of DIAP1 induced rapid apoptosis in these cells, whereas the inhibition of DIAP2 expression did not but resulted in increased sensitivity to stress-induced apoptosis; apoptosis in both cases was prevented by inhibition of ARK expression. Cells in which cytochrome c expression was decreased underwent apoptosis induced by stress stimuli, Reaper or Grim. These results demonstrate the central role of ARK in stress-induced apoptosis, which appears to act independently of cytochrome c. Apoptosis induced by Reaper or Grim can proceed via a distinct pathway, independent of ARK. PMID:11901172

Heterogeneous nuclear ribonucleoprotein H Blocks MST2-Dependent Apoptosis in Cancer Cells via Regulation of A-Raf transcription

PubMed Central

Rauch, Jens; O'Neill, Eric; Mack, Brigitte; Matthias, Christoph; Munz, Markus; Kolch, Walter; Gires, Olivier

2010-01-01

Summary A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signalling by activating the MEK-ERK pathway. Low kinase activity of A-Raf towards MEK suggested that A-Raf might have alternative functions. We show that A-Raf prevents cancer cell apoptosis contingent on the expression of the hnRNP H splice factor, which is required for the correct transcription and expression of A-Raf. A-Raf prevented apoptosis by sequestering and inactivating the pro-apoptotic MST2 kinase. Knock-down of hnRNP H or A-Raf resulted in MST2-dependent apoptosis, while enforced expression of either one partially counteracted apoptosis induced by etoposide. In vivo expression studies in colon specimens corroborated the over-expression of hnRNP H in malignant tissues and its correlation with A-Raf levels. In summary, we present a novel route that is usurped by tumor cells to escape naturally imposed apoptotic signals. PMID:20145135

Expression of Bcl-2 and NF-κB in brain tissue after acute renal ischemia-reperfusion in rats.

PubMed

Zhang, Na; Cheng, Gen-Yang; Liu, Xian-Zhi; Zhang, Feng-Jiang

2014-05-01

To investigate the effect of acute renal ischemia reperfusion on brain tissue. Fourty eight rats were randomly divided into four groups (n=12): sham operation group, 30 min ischemia 60 min reperfusion group, 60 min ischemia 60 min reperfusion group, and 120 min ischemia 60 min reperfusion group. The brain tissues were taken after the experiment. TUNEL assay was used to detect the brain cell apoptosis, and western blot was used to detect the expression of apoptosis-related proteins and inflammatory factors. Renal ischemia-reperfusion induced apoptosis of brain tissues, and the apoptosis increased with prolongation of ischemia time. The detection at the molecular level showed decreased Bcl-2 expression, increased Bax expression, upregulated expression of NF-κB and its downstream factor COX-2/PGE2. Acute renal ischemia-reperfusion can cause brain tissue damage, manifested as induced brain tissues apoptosis and inflammation activation. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

Neuroprotective effects of ganoderma lucidum polysaccharides against oxidative stress-induced neuronal apoptosis.

PubMed

Sun, Xin-Zhi; Liao, Ying; Li, Wei; Guo, Li-Mei

2017-06-01

Ganoderma lucidum polysaccharides have protective effects against apoptosis in neurons exposed to ischemia/reperfusion injury, but the mechanisms are unclear. The goal of this study was to investigate the underlying mechanisms of the effects of ganoderma lucidum polysaccharides against oxidative stress-induced neuronal apoptosis. Hydrogen peroxide (H 2 O 2 ) was used to induce apoptosis in cultured cerebellar granule cells. In these cells, ganoderma lucidum polysaccharides remarkably suppressed H 2 O 2 -induced apoptosis, decreased expression of caspase-3, Bax and Bim and increased that of Bcl-2. These findings suggested that ganoderma lucidum polysaccharides regulate expression of apoptosis-associated proteins, inhibit oxidative stress-induced neuronal apoptosis and, therefore, have significant neuroprotective effects.

Cigarette smoking induces heat shock protein 70 kDa expression and apoptosis in rat brain: Modulation by bacoside A.

PubMed

Anbarasi, K; Kathirvel, G; Vani, G; Jayaraman, G; Shyamala Devi, C S

2006-01-01

Cigarette smoking is associated with the development of several diseases and antioxidants play a major role in the prevention of smoking-related diseases. Apoptosis is suggested as a possible contributing factor in the pathogenesis of smoking-induced toxicity. Therefore the present study was designed to investigate the influence of chronic cigarette smoke exposure on apoptosis and the modulatory effect of bacoside A (triterpenoid saponin isolated from the plant Bacopa monniera) on smoking-induced apoptosis in rat brain. Adult male albino rats of Wistar strain were exposed to cigarette smoke and simultaneously administered with bacoside A (10 mg/kg b.w./day, orally) for a period of 12 weeks. Expression of brain hsp70 was analyzed by Western blotting. Apoptosis was identified by DNA fragmentation, terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick end labeling (TUNEL) staining and transmission electron microscopy. The results showed that exposure to cigarette smoke induced hsp70 expression and apoptosis as characterized by DNA laddering, increased TUNEL-positive cells and ultrastructural apoptotic features in the brain. Administration of bacoside A prevented expression of hsp70 and neuronal apoptosis during cigarette smoking. We speculate that apoptosis may be responsible for the smoking-induced brain damage and bacoside A can protect the brain from the toxic effects of cigarette smoking.

Proteomics Identification of Annexin A2 as a Key Mediator in the Metastasis and Proangiogenesis of Endometrial Cells in Human Adenomyosis*

PubMed Central

Zhou, Shengtao; Yi, Tao; Liu, Rui; Bian, Ce; Qi, Xiaorong; He, Xiang; Wang, Kui; Li, Jingyi; Zhao, Xia; Huang, Canhua; Wei, Yuquan

2012-01-01

Adenomyosis is a common estrogen-dependent disorder of females characterized by a downward extension of the endometrium into the uterine myometrium and neovascularization in ectopic lesions. It accounts for chronic pelvic pain, dysmenorrhea, menorrhagia, and infertility in 8.8–61.5% women worldwide. However, the molecular mechanisms for adenomyosis development remain poorly elucidated. Here, we utilized a two-dimensional polyacrylamide gel electrophoresis/MS-based proteomics analysis to compare and identify differentially expressed proteins in matched ectopic and eutopic endometrium of adenomyosis patients. A total of 93 significantly altered proteins were identified by tandem MS analysis. Further cluster analysis revealed a group of estrogen-responsive proteins as dysregulated in adenomyosis, among which annexin A2, a member of annexin family proteins, was found up-regulated most significantly in the ectopic endometrium of adenomyosis compared with its eutopic counterpart. Overexpression of ANXA2 was validated in ectopic lesions of human adenomyosis and was found to be tightly correlated with markers of epithelial to mesenchymal transition and dysmenorrhea severity of adenomyosis patients. Functional analysis demonstrated that estrogen could remarkably up-regulate ANXA2 and induce epithelial to mesenchymal transition in an in vitro adenomyosis model. Enforced expression of ANXA2 could mediate phenotypic mesenchymal-like cellular changes, with structural and functional alterations in a β-catenin/T-cell factor (Tcf) signaling-associated manner, which could be reversed by inhibition of ANXA2 expression. We also proved that enforced expression of ANXA2 enhanced the proangiogenic capacity of adenomyotic endometrial cells through HIF-1α/VEGF-A pathway. In vivo, we demonstrated that ANXA2 inhibition abrogated endometrial tissue growth, metastasis, and angiogenesis in an adenomyosis nude mice model and significantly alleviated hyperalgesia. Taken together, our data unraveled a dual role for ANXA2 in the pathogenesis of human adenomyosis through conferring endometrial cells both metastatic potential and proangiogenic capacity, which could serve as a potential therapeutic target for the treatment of adenomyosis patients. PMID:22493182

The evolutionary duplication and probable demise of an endodermal GATA factor in Caenorhabditis elegans.

PubMed

Fukushige, Tetsunari; Goszczynski, Barbara; Tian, Helen; McGhee, James D

2003-10-01

We describe the elt-4 gene from the nematode Caenorhabditis elegans. elt-4 is predicted to encode a very small (72 residues, 8.1 kD) GATA-type zinc finger transcription factor. The elt-4 gene is located approximately 5 kb upstream of the C. elegans elt-2 gene, which also encodes a GATA-type transcription factor; the zinc finger DNA-binding domains are highly conserved (24/25 residues) between the two proteins. The elt-2 gene is expressed only in the intestine and is essential for normal intestinal development. This article explores whether elt-4 also has a role in intestinal development. Reporter fusions to the elt-4 promoter or reporter insertions into the elt-4 coding regions show that elt-4 is indeed expressed in the intestine, beginning at the 1.5-fold stage of embryogenesis and continuing into adulthood. elt-4 reporter fusions are also expressed in nine cells of the posterior pharynx. Ectopic expression of elt-4 cDNA within the embryo does not cause detectable ectopic expression of biochemical markers of gut differentiation; furthermore, ectopic elt-4 expression neither inhibits nor enhances the ectopic marker expression caused by ectopic elt-2 expression. A deletion allele of elt-4 was isolated but no obvious phenotype could be detected, either in the gut or elsewhere; brood sizes, hatching efficiencies, and growth rates were indistinguishable from wild type. We found no evidence that elt-4 provided backup functions for elt-2. We used microarray analysis to search for genes that might be differentially expressed between L1 larvae of the elt-4 deletion strain and wild-type worms. Paired hybridizations were repeated seven times, allowing us to conclude, with some confidence, that no candidate target transcript could be identified as significantly up- or downregulated by loss of elt-4 function. In vitro binding experiments could not detect specific binding of ELT-4 protein to candidate binding sites (double-stranded oligonucleotides containing single or multiple WGATAR sequences); ELT-4 protein neither enhanced nor inhibited the strong sequence-specific binding of the ELT-2 protein. Whereas ELT-2 protein is a strong transcriptional activator in yeast, ELT-4 protein has no such activity under similar conditions, nor does it influence the transcriptional activity of coexpressed ELT-2 protein. Although an elt-2 homolog was easily identified in the genomic sequence of the related nematode C. briggsae, no elt-4 homolog could be identified. Analysis of the changes in silent third codon positions within the DNA-binding domains indicates that elt-4 arose as a duplication of elt-2, some 25-55 MYA. Thus, elt-4 has survived far longer than the average duplicated gene in C. elegans, even though no obvious biological function could be detected. elt-4 provides an interesting example of a tandemly duplicated gene that may originally have been the same size as elt-2 but has gradually been whittled down to its present size of little more than a zinc finger. Although elt-4 must confer (or must have conferred) some selective advantage to C. elegans, we suggest that its ultimate evolutionary fate will be disappearance from the C. elegans genome.

Genetic Ablation of the Aryl Hydrocarbon Receptor Causes Cigarette Smoke-induced Mitochondrial Dysfunction and Apoptosis*

PubMed Central

Rico de Souza, Angela; Zago, Michela; Pollock, Stephen J.; Sime, Patricia J.; Phipps, Richard P.; Baglole, Carolyn J.

2011-01-01

Cigarette smoke is the primary risk factor for chronic obstructive pulmonary disease (COPD). Alterations in the balance between apoptosis and proliferation are involved in the etiology of COPD. Fibroblasts and epithelial cells are sensitive to the oxidative properties of cigarette smoke, and whose loss may precipitate the development of COPD. Fibroblasts express the aryl hydrocarbon receptor (AhR), a transcription factor that attenuates pulmonary inflammation and may also regulate apoptosis. We hypothesized the AhR would prevent apoptosis caused by cigarette smoke. Using genetically deleted in vitro AhR expression models and an established method of cigarette smoke exposure, we report that AhR expression regulates fibroblasts proliferation and prevents morphological features of apoptosis, including membrane blebbing and chromatin condensation caused by cigarette smoke extract (CSE). Absence of AhR expression results in cleavage of PARP, lamin, and caspase-3. Mitochondrial dysfunction, including cytochrome c release, was associated with loss of AhR expression, indicating activation of the intrinsic apoptotic cascade. Heightened sensitivity of AhR-deficient fibroblasts was not the result of alterations in GSH, Nrf2, or HO-1 expression. Instead, AhR−/− cells had significantly less MnSOD and CuZn-SOD expression, enzymes that protects against oxidative stress. The ability of the AhR to suppress apoptosis was not restricted to fibroblasts, as siRNA-mediated knockdown of the AhR in lung epithelial cells also increased sensitivity to smoke-induced apoptosis. Collectively, these results suggest that cigarette smoke induced loss of lung structural support (i.e. fibroblasts, epithelial cells) caused by aberrations in AhR expression may explain why some smokers develop lung diseases such as COPD. PMID:21984831

Protective role of Osthole on myocardial cell apoptosis induced by doxorubicin in rats.

PubMed

Xu, Hongdang; Han, Yu; Zhang, Mengwei; Yan, Min; Gao, Chuanyu

2015-01-01

To explore the effect of Osthole on protecting myocardial cell apoptosis induced by doxorubicin during cardiac failure in rats. Myocardial cells isolated from the newborn SD rats were separated into three groups: cells treated with 1 μmol doxorubicin, cells treated with Osthole at three concentrations of 10, 20, and 40 μmol, cells treated neither with Osthole nor with doxorubicin were the control groups. Consequently, cell apoptosis of myocardial cells in each group was analyzed using TUNEL assay. Also, expressions of oxidase, NADPH, and ROS in myocardial cells were analyzed using different biological methods. Moreover, expressions of cell apoptosis associated proteins were analyzed using Western blotting. Compared with the controls, the results showed that cells received Osthole and doxorubicin treatments performed high percentage of cell apoptosis, suggesting that Osthole could anesis myocardial cell apoptosis induced by doxorubicin (P<0.05). Osthole of 10 μmol depressed the expressions of cell apoptosis associated proteins including Caspase-3 and Cytc, and enhancing expression of Bcl-XL expression (P<0.05). Osthole of 20 μmol significantly decreased the generation of intracellar superoxidase, NADPH, and NADPH activity in myocardial cells treated with doxorubicin (P<0.05). Moreover, Osthole of 20 μmol could significantly increase phosphorylated elF2α level in cells. Our study suggested that Osthole may play a protective role in suppressing myocardial apoptosis induced by doxorubicin through inhibiting NADPH and superoxidase production and downstream phosphorylated elF2α.

Protective role of Osthole on myocardial cell apoptosis induced by doxorubicin in rats

PubMed Central

Xu, Hongdang; Han, Yu; Zhang, Mengwei; Yan, Min; Gao, Chuanyu

2015-01-01

Objective: To explore the effect of Osthole on protecting myocardial cell apoptosis induced by doxorubicin during cardiac failure in rats. Methods: Myocardial cells isolated from the newborn SD rats were separated into three groups: cells treated with 1 μmol doxorubicin, cells treated with Osthole at three concentrations of 10, 20, and 40 μmol, cells treated neither with Osthole nor with doxorubicin were the control groups. Consequently, cell apoptosis of myocardial cells in each group was analyzed using TUNEL assay. Also, expressions of oxidase, NADPH, and ROS in myocardial cells were analyzed using different biological methods. Moreover, expressions of cell apoptosis associated proteins were analyzed using Western blotting. Results: Compared with the controls, the results showed that cells received Osthole and doxorubicin treatments performed high percentage of cell apoptosis, suggesting that Osthole could anesis myocardial cell apoptosis induced by doxorubicin (P<0.05). Osthole of 10 μmol depressed the expressions of cell apoptosis associated proteins including Caspase-3 and Cytc, and enhancing expression of Bcl-XL expression (P<0.05). Osthole of 20 μmol significantly decreased the generation of intracellar superoxidase, NADPH, and NADPH activity in myocardial cells treated with doxorubicin (P<0.05). Moreover, Osthole of 20 μmol could significantly increase phosphorylated elF2α level in cells. Conclusion: Our study suggested that Osthole may play a protective role in suppressing myocardial apoptosis induced by doxorubicin through inhibiting NADPH and superoxidase production and downstream phosphorylated elF2α. PMID:26617794

Activator protein 1 promotes gemcitabine-induced apoptosis in pancreatic cancer by upregulating its downstream target Bim.

PubMed

Ren, Xiaoxia; Zhao, Wenjing; Du, Yongxing; Zhang, Taiping; You, Lei; Zhao, Yupei

2016-12-01

Gemcitabine is a commonly used chemotherapy drug in pancreatic cancer. The function of activator protein 1 (AP-1) is cell-specific, and its function depends on the expression of other complex members. In the present study, we added gemcitabine to the media of Panc-1 and SW1990 cells at clinically achieved concentrations (10 µM). Compared with constitutive c-Fos expression, c-Jun expression increased in a dose-dependent manner upon gemcitabine treatment. c-Jun overexpression increased gemcitabine-induced apoptosis through Bim activation, while cell apoptosis and Bim expression decreased following c-Jun knockdown. Furthermore, gemcitabine-induced apoptosis and Bim levels decreased when c-Jun phosphorylation was blocked by SP600125. Our findings suggest that c-Jun, which is a member of the AP-1 complex, functions in gemcitabine-induced apoptosis by regulating its downstream target Bim in pancreatic cancer cells.

MicroRNA-15b deteriorates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by downregulating Bcl-2 and MAPK3.

PubMed

Liu, Yaling; Yang, Liqun; Yin, Jiemin; Su, Diansan; Pan, Zhiying; Li, Peiying; Wang, Xiaodong

2018-01-01

To investigate the role of miRNA-15b in cardiomyocyte apoptosis after ischemia reperfusion injury in acute myocardial infarction (AMI), we conducted the AMI rat model by using left anterior descending ligation and performed hypoxia/reoxygenation experiments in H9c2 cells. MiRNA-15b was measured by quantitative reverse transcription PCR (qRT-PCR). Cardiomyocyte apoptosis was determined by terminal deoxynucleotide transferase dUTP nick end labeling staining. Synthesized miRNA-15b mimic and inhibitor were transfected into H9c2 cells by Lipofectamine regent. RNA expression of B cell lymphoma/leukemia-2 (Bcl-2) and mitogen-activated protein kinase 3 (MAPK3) was examined by qRT-PCR and their protein expression was determined by western blot. Ischemia reperfusion increased miRNA-15b expression in the ischemic rat heart and resulted more severe cardiomyocytes apoptosis. In H9c2 cells, hypoxia/reoxygenation induced increased miRNA-15b expression and augmented cardiomyocyte apoptosis observed at 24 hours after 24-hour hypoxia. Compared with the vehicle group, miRNA-15b mimic further raised miRNA-15b level and increased cardiomyocyte apoptosis, whereas miRNA-15b inhibitor suppressed miRNA-15b expression and protected cardiomyocytes from apoptosis. Although the mRNA expression of the target genes Bcl-2 and MAPK3 was not changed significantly, the protein expression of these two genes were markedly reduced after miRNA-15b mimic treatment and significantly increased after transfected with miRNA-15b inhibitors. In conclusion, miRNA-15b deteriorates cardiomyocyte apoptosis by post-transcriptionally downregulating the expression of Bcl-2 and MAPK3. © American Federation for Medical Research (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium.

PubMed

Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J; Klein, Ophir D; Barlow, Linda A

2014-08-01

Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation. © 2014. Published by The Company of Biologists Ltd.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A)-Mediated changes in Fas Expression and Fas-Dependent Apoptosis: Role of Lyn/Syk activation

PubMed Central

Incrocci, Ryan; Hussain, Samira; Stone, Amanda; Bieging, Kathryn; Alt, Lauren A.C.; Fay, Michael J.; Swanson-Mungerson, Michelle

2015-01-01

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies. PMID:26255694

Apoptosis induced by low-level laser in polymorphonuclear cells of acute joint inflammation: comparative analysis of two energy densities.

PubMed

Dos Anjos, Lúcia Mara Januário; da Fonseca, Adenilson de Souza; Gameiro, Jacy; de Paoli, Flávia

2017-07-01

Anti-inflammatory property of low-level laser therapy (LLLT) has been widely described in literature, although action mechanisms are not always clarified. Thus, this study aimed to evaluate apoptosis mechanisms in the LLLT anti-inflammatory effects on the arthritis experimental model in vivo at two different energy densities (3 and 30 Jcm -2 ). Arthritis was induced in mice by zymosan solution, animals were distributed into five groups, and morphological analysis, immunocytochemistry and gene expressions for apoptotic proteins were performed. Data showed an anti-inflammatory effect, DNA fragmentation in polymorphonuclear (PMN) cells and alteration in gene expression of proteins related to apoptosis pathways after LLLT. p53 gene expression increased at both energy densities, Bcl2 gene expression increased at 3 Jcm -2 , and Bcl2 tissue expression decreased at 30 Jcm -2 . In addition, apoptosis was restricted to PMN cells. Results suggest that apoptosis in PMN cells comprise part of LLLT anti-inflammatory mechanisms by disbalance promotion between expression of pro-apoptotic (Bax and p53) and anti-apoptotic (Bcl-2) proteins, with pro-apoptotic gene expression selectively in PMN cells.

Effect of curcumin on pathogenesis of hamster-opisthorchiasis through apoptosis-related gene expression.

PubMed

Sriraj, Pranee; Boonmars, Thidarut; Boonjaraspinyo, Sirintip; Kaewsamut, Butsara; Srisawangwong, Tuanchai; Sithithaworn, Paiboon; Wu, Zhiliang

2009-11-01

The present study investigated the effect of curcumin, a phenolic compound with yellow color from Curcuma longa L., on the expression of the apoptosis-related genes [BAX (Bcl-2 associated protein X), PKB, p53, MDM2 (mouse double minute 2), caspase 9, c-Ski, smad1 and smad4] in hamster opisthorchiasis. On Opisthorchis viverrini infection treated with dietary curcumin apoptosis-related gene expression profiles were similar to O. viverrini-infected group, but the expression levels seemed lower. Light microscopic observation revealed that aggregation of inflammatory cells surrounding the hepatic bile ducts in the groups infected with O. viverrini and treated with dietary curcumin was lower than in infected group. The intensity of the response is correlated with expression of the genes studied. The results suggest that curcumin reduces pathogenesis in hamster-opisthorchiasis by controlling apoptosis-related gene expression.

Dietary effects of cotton tissue expressing germin like protein on beet armyworm (Lepidoptera: Noctuidae) growth, survival and pupation

USDA-ARS?s Scientific Manuscript database

Transgenic cotton lines that ectopically express a cotton germin-like protein (ABP) were screened for resistance/tolerance factors to the beet armyworm (BAW) Spodoptera exigua (Hubner) via feeding assays. The number of BAW eggs that successfully hatched was not statistically different at 72 h observ...

Differential effects on apoptosis induction in hepatocyte lines by stable expression of hepatitis B virus X protein

PubMed Central

Fiedler, Nicola; Quant, Ellen; Fink, Ludger; Sun, Jianguang; Schuster, Ralph; Gerlich, Wolfram H; Schaefer, Stephan

2006-01-01

AIM: Hepatitis B virus protein X (HBx) has been shown to be weakly oncogenic in vitro. The transforming activities of HBx have been linked with the inhibition of several functions of the tumor suppressor p53. We have studied whether HBx may have different effects on p53 depending on the cell type. METHODS: We used the human hepatoma cell line HepG2 and the immortalized murine hepatocyte line AML12 and analyzed stably transfected clones which expressed physiological amounts of HBx. P53 was induced by UV irradiation. RESULTS: The p53 induction by UV irradiation was unaffected by stable expression of HBx. However, the expression of the cyclin kinase inhibitor p21waf/cip/sdi which gets activated by p53 was affected in the HBx transformed cell line AML12-HBx9, but not in HepG2. In AML-HBx9 cells, p21waf/cip/sdi-protein expression and p21waf/cip/sdi transcription were deregulated. Furthermore, the process of apoptosis was affected in opposite ways in the two cell lines investigated. While stable expression of HBx enhanced apoptosis induced by UV irradiation in HepG2-cells, apoptosis was decreased in HBx transformed AML12-HBx9. P53 repressed transcription from the HBV enhancer I, when expressed from expression vectors or after induction of endogenous p53 by UV irradiation. Repression by endogenous p53 was partially reversible by stably expressed HBx in both cell lines. CONCLUSION: Stable expression of HBx leads to deregulation of apoptosis induced by UV irradiation depending on the cell line used. In an immortalized hepatocyte line HBx acted anti-apoptotic whereas expression in a carcinoma derived hepatocyte line HBx enhanced apoptosis. PMID:16937438

Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice.

PubMed

Wolff, G L; Kodell, R L; Moore, S R; Cooney, C A

1998-08-01

'Viable yellow' (Avy/a) mice are larger, obese, hyperinsulinemic, more susceptible to cancer, and, on average, shorter lived than their non-yellow siblings. They are epigenetic mosaics ranging from a yellow phenotype with maximum ectopic agouti overexpression, through a continuum of mottled agouti/yellow phenotypes with partial agouti overexpression, to a pseudoagouti phenotype with minimal ectopic expression. Pseudoagouti Avy/a mice are lean, healthy, and longer lived than their yellow siblings. Here we report that feeding pregnant black a/a dams methyl-supplemented diets alters epigenetic regulation of agouti expression in their offspring, as indicated by increased agouti/black mottling in the direction of the pseudoagouti phenotype. We also present confirmatory evidence that epigenetic phenotypes are maternally heritable. Thus Avy expression, already known to be modulated by imprinting, strain-specific modification, and maternal epigenetic inheritance, is also modulated by maternal diet. These observations suggest, at least in this special case, that maternal dietary supplementation may positively affect health and longevity of the offspring. Therefore, this experimental system should be useful for identifying maternal factors that modulate epigenetic mechanisms, especially DNA methylation, in developing embryos.

Expression of motility-related molecule Cdc42 in endometrial tissue in women with adenomyosis and ovarian endometriomata.

PubMed

Goteri, Gaia; Ciavattini, Andrea; Lucarini, Guendalina; Montik, Nina; Filosa, Alessandra; Stramazzotti, Daniela; Biagini, Graziella; Tranquilli, Andrea Luigi

2006-09-01

To evaluate Cdc42 expression in eutopic and ectopic endometrial tissue in patients with adenomyosis and ovarian endometriotic cysts compared with patients without endometriosis. Experimental retrospective study. University hospital. Twenty-four patients with adenomyosis, 19 with ovarian endometriomata, and 9 with fibroids or benign ovarian cysts. Hysterectomy and bilateral oophorectomy. Immunostaining for Cdc42 of eutopic and ectopic endometrial tissues. In eutopic endometrium of patients with adenomyosis and with fibroids or benign ovarian cysts, the intensity of Cdc42 immunostaining was weaker, especially in the specialized stromal cells, compared with cases with ovarian endometriosis (chi(2) test, P=.003). Expression of Cdc42 in eutopic endometrium showed a trend to be higher in the secretory than in the proliferative phase and in patients with ovarian endometriotic cysts compared with patients with adenomyosis (unpaired t test, P=.005), especially in the proliferative phase. An abnormally high expression of Cdc42 in eutopic endometrium in the secretory phase may contribute to the development of ovarian endometriosis, but it does not seem to be involved in the pathogenesis of adenomyosis.

Altered serotonin physiology in human breast cancers favors paradoxical growth and cell survival.

PubMed

Pai, Vaibhav P; Marshall, Aaron M; Hernandez, Laura L; Buckley, Arthur R; Horseman, Nelson D

2009-01-01

The breast microenvironment can either retard or accelerate the events associated with progression of latent cancers. However, the actions of local physiological mediators in the context of breast cancers are poorly understood. Serotonin (5-HT) is a critical local regulator of epithelial homeostasis in the breast and other organs. Herein, we report complex alterations in the intrinsic mammary gland serotonin system of human breast cancers. Serotonin biosynthetic capacity was analyzed in human breast tumor tissue microarrays using immunohistochemistry for tryptophan hydroxylase 1 (TPH1). Serotonin receptors (5-HT1-7) were analyzed in human breast tumors using the Oncomine database. Serotonin receptor expression, signal transduction, and 5-HT effects on breast cancer cell phenotype were compared in non-transformed and transformed human breast cells. In the context of the normal mammary gland, 5-HT acts as a physiological regulator of lactation and involution, in part by favoring growth arrest and cell death. This tightly regulated 5-HT system is subverted in multiple ways in human breast cancers. Specifically, TPH1 expression undergoes a non-linear change during progression, with increased expression during malignant progression. Correspondingly, the tightly regulated pattern of 5-HT receptors becomes dysregulated in human breast cancer cells, resulting in both ectopic expression of some isoforms and suppression of others. The receptor expression change is accompanied by altered downstream signaling of 5-HT receptors in human breast cancer cells, resulting in resistance to 5-HT-induced apoptosis, and stimulated proliferation. Our data constitutes the first report of direct involvement of 5-HT in human breast cancer. Increased 5-HT biosynthetic capacity accompanied by multiple changes in 5-HT receptor expression and signaling favor malignant progression of human breast cancer cells (for example, stimulated proliferation, inappropriate cell survival). This occurs through uncoupling of serotonin from the homeostatic regulatory mechanisms of the normal mammary epithelium. The findings open a new avenue for identification of diagnostic and prognostic markers, and valuable new therapeutic targets for managing breast cancer.

The Toxoplasma gondii ME-49 strain upregulates levels of A20 that inhibit NF-κB activation and promotes apoptosis in human leukaemia T-cell lines.

PubMed

Chen, Qian; Pang, Min-Hui; Ye, Xiao-Hong; Yang, Guang; Lin, Chen

2018-05-18

Acute T-lymphocyte leukaemia is a form of haematological malignancy with abnormal activation of NF-κB pathway, which results in high expression of A20 and ABIN1, which constitute a negative feedback mechanism for the regulation of NF-κB activation. Clinical studies have found that acute T-lymphocyte leukaemia patients are susceptible to Toxoplasma gondii infection; however, the effect of T. gondii on the proliferation and apoptosis of human leukaemia T-cells remains unclear. Here, we used the T. gondii ME-49 strain to infect human leukaemia T-cell lines Jurkat and Molt-4, to explore the effect of T. gondii on proliferation and apoptosis, which is mediated by NF-κB in human leukaemia T-cells. The Tunel assay was used to detect cell apoptosis. Cell Counting Kit-8 was used to detect cell proliferation viability. The apoptosis level and the expression level of NF-κB related proteins in human leukaemia T-cells were detected by flow cytometry and Western blotting. Western blotting analyses revealed that the T. gondii ME-49 strain increased the expression of A20 and decreased both ABIN1 expression and NF-κB p65 phosphorylation. By constructing a lentiviral-mediated shRNA to knockdown the A20 gene in Jurkat T-cells and Molt-4 T-cells, the apoptosis levels of the two cell lines decreased after T. gondii ME-49 infection, and levels of NF-κB p65 phosphorylation and ABIN1 were higher than in the non-konckdown group. After knockingdown ABIN1 gene expression by constructing the lentiviral-mediated shRNA and transfecting the recombinant expression plasmid containing the ABIN1 gene into two cell lines, apoptosis levels and cleaved caspase-8 expression increased or decreased in response to T. gondii ME-49 infection, respectively. Our data suggest that ABIN1 protects human leukaemia T-cells by allowing them to resist the apoptosis induced by T. gondii ME-49 and that the T. gondii ME-49 strain induces the apoptosis of human leukaemia T-cells via A20-mediated downregulation of ABIN1 expression.

Differential effects of alloherpesvirus CyHV-3 and rhabdovirus SVCV on apoptosis in fish cells.

PubMed

Miest, Joanna J; Adamek, Mikolaj; Pionnier, Nicolas; Harris, Sarah; Matras, Marek; Rakus, Krzysztof Ł; Irnazarow, Ilgiz; Steinhagen, Dieter; Hoole, Dave

2015-03-23

Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process. Copyright © 2015 Elsevier B.V. All rights reserved.

mir-200c Regulates Induction of Apoptosis through CD95 by Targeting FAP-1

PubMed Central

Schickel, Robert; Park, Sun-Mi; Murmann, Andrea E.; Peter, Marcus E.

2010-01-01

SUMMARY Tumor progression shares many characteristics with the process of epithelial-to-mesenchymal transition (EMT). Cells that have undergone an EMT are known to have an increased resistance to apoptosis. CD95/Fas is an apoptosis-inducing receptor expressed on many tissues and tumor cells. During tumor progression CD95 is frequently downregulated, and tumor cells lose apoptosis sensitivity. miR-200 microRNAs repress both the EMT-inducing ZEB1 and ZEB2 transcription factors. We now demonstrate that miR-200c sensitizes cells to apoptosis mediated by CD95. We have identified the apoptosis inhibitor FAP-1 as a target for miR-200c. FAP-1 was demonstrated to be responsible for the reduced sensitivity to CD95-mediated apoptosis in cells with inhibited miR-200. The identification of FAP-1 as a miR-200c target provides a molecular mechanism to explain both the downregulation of CD95 expression and the reduction in sensitivity of cells to CD95-mediated apoptosis that is observed in the context of reduced miR-200 expression during tumor progression. PMID:20620960

Lack of induction of tissue transglutaminase but activation of the preexisting enzyme in c-Myc-induced apoptosis of CHO cells.

PubMed

Balajthy, Z; Kedei, N; Nagy, L; Davies, P J; Fésüs, L

1997-07-18

The intracellular activity and expression of tissue transglutaminase, which crosslinks proteins through epsilon(gamma-glutamyl)lysine isodipeptide bond, was investigated in CHO cells and those stably transfected with either inducible c-Myc (which leads to apoptosis) or with c-myc and the apoptosis inhibitor Bcl-2. Protein-bound cross-link content was significantly higher when apoptosis was induced by c-Myc while the concomitant presence of Bcl-2 markedly reduced both apoptosis and enzymatic protein cross-linking. The expression of tissue transglutaminase did not change following the initiation of apoptosis by c-Myc or when it was blocked by Bcl-2. Studying transiently co-transfected elements of the mouse tissue transglutaminase promoter linked to a reporter enzyme revealed their overall repression in cells expressing c-Myc. This repression was partially suspended in cells also carrying Bcl-2. Our data suggest that tissue transglutaminase is not induced when c-Myc initiates apoptosis but the pre-existing endogenous enzyme is activated.

Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression*

PubMed Central

Wang, Chunmei; Qi, Runzi; Li, Nan; Wang, Zhengxin; An, Huazhang; Zhang, Qinghua; Yu, Yizhi; Cao, Xuetao

2009-01-01

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC. PMID:19376776

Cancer-selective death of human breast cancer cells by leelamine is mediated by bax and bak activation.

PubMed

Sehrawat, Anuradha; Kim, Su-Hyeong; Hahm, Eun-Ryeong; Arlotti, Julie A; Eiseman, Julie; Shiva, Sruti S; Rigatti, Lora H; Singh, Shivendra V

2017-02-01

The present study is the first to report inhibition of breast cancer cell growth in vitro and in vivo and suppression of self-renewal of breast cancer stem cells (bCSC) by a pine bark component (leelamine). Except for a few recent publications in melanoma, anticancer pharmacology of this interesting phytochemical is largely elusive. Leelamine (LLM) dose-dependently inhibited viability of MDA-MB-231 (triple-negative), MCF-7 (estrogen receptor-positive), and SUM159 (triple-negative) human breast cancer cells in association with apoptotic cell death induction. To the contrary, a normal mammary epithelial cell line derived from fibrocystic breast disease and spontaneously immortalized (MCF-10A) was fully resistant to LLM-mediated cell growth inhibition and apoptosis induction. LLM also inhibited self-renewal of breast cancer stem cells. Apoptosis induction by LLM in breast cancer cells was accompanied by a modest increase in reactive oxygen species production, which was not due to inhibition of mitochondrial electron transport chain complexes. Nevertheless, ectopic expression of manganese superoxide dismutase conferred partial protection against LLM-induced cell death but only at a lower yet pharmacologically relevant concentration. Exposure of breast cancer cells to LLM resulted in (a) induction and/or activation of multidomain proapoptotic proteins Bax and Bak, (b) caspase-9 activation, and (c) cytosolic release of cytochrome c. Bax and Bak deficiency in immortalized fibroblasts conferred significant protection against cell death by LLM. Intraperitoneal administration of LLM (7.5 mg/kg; 5 times/wk) suppressed the growth of orthotopic SUM159 xenografts in mice without any toxicity. In conclusion, the present study provides critical preclinical data to warrant further investigation of LLM. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Induction of E-cadherin in lung cancer and interaction with growth suppression by histone deacetylase inhibition.

PubMed

Kakihana, Masatoshi; Ohira, Tatsuo; Chan, Daniel; Webster, Robin B; Kato, Harubumi; Drabkin, Harry A; Gemmill, Robert M

2009-12-01

Loss of E-cadherin confers a poor prognosis in lung cancer patients and is associated with in vitro resistance to endothelial growth factor receptor inhibitors. Zinc finger E box-binding homeobox (ZEB)-1, the predominant transcriptional suppressor of E-cadherin in lung tumor lines, recruits histone deacetylases (HDACs) as co-repressors. NSCLC cell lines were treated with HDAC inhibitors and analyzed for E-cadherin induction, growth inhibition and apoptosis. National Cancer Institute-H157 cells expressing ectopic E-cadherin were tested for tumorigenicity in murine xenografts. We found that treatment with MS-275, compared to vorinostat (SAHA), valproic acid or trichostatin A, was most effective in E-cadherin up-regulation and persistence in non-small cell lung cancers. As with other tumor types and HDAC inhibitors, MS-275 inhibited growth and induced apoptosis. Importantly, blocking E-cadherin induction by short hairpin RNA resulted in less inhibition by MS-275, implicating the epithelial to mesenchymal phenotype process as a contributing factor. In contrast to H460 and H661, H157 cells were resistant to E-cadherin up-regulation by HDAC inhibitors. However, E-cadherin was restored, in a synergistic manner, by combined knockdown of ZEB-1 and ZEB-2. In addition, H157 cells stably transfected with E-cadherin were markedly attenuated in their tumor forming ability. Lastly, combining MS-275 with the microtubule stabilizing agent, paclitaxel, or 17-(allylamino)-17-demethoxygeldanamycin, a heat shock protein 90 inhibitor, resulted in synergistic growth inhibition. Since MS-275 has no reported activity against HDAC6, which regulates both microtubule and heat shock protein 90 functions, other mechanisms of synergy are anticipated. These results support the role of ZEB proteins and HDAC inhibitors in the pathogenesis and treatment of lung cancer.

Novel mechanisms of early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice

PubMed Central

Hoshi, Masato; Batourina, Ekatherina; Mendelsohn, Cathy; Jain, Sanjay

2012-01-01

Mutations in the receptor tyrosine kinase RET are associated with congenital anomalies of kidneys or urinary tract (CAKUT). RET tyrosine Y1015 is the docking site for PLCγ, a major regulator of RET signaling. Abrogating signaling via Y1015 causes CAKUT that are markedly different than renal agenesis in Ret-null or RetY1062F mutant mice. We performed analysis of Y1015F mutant upper and lower urinary tracts in mice to delineate its molecular and developmental roles during early urinary tract formation. We found that the degeneration of the common nephric ducts (CND), the caudal-most Wolffian duct (WD) segment, depends on Y1015 signals. The CNDs in Y1015F mutants persist owing to increased proliferation and reduced apoptosis, and showed abundance of phospho-ERK-positive cells. In the upper urinary tract, the Y1015 signals are required for proper patterning of the mesonephros and metanephros. Timely regression of mesonephric mesenchyme and proper demarcation of mesonephric and metanephric mesenchyme from the WD depends on RetY1015 signaling. We show that the mechanism of de novo ectopic budding is via increased ERK activity due to abnormal mesenchymal GDNF expression. Although reduction in GDNF dosage improved CAKUT it did not affect delayed mesenchyme regression. Experiments using whole-mount immunofluorescence confocal microscopy and explants cultures of early embryos with ERK-specific inhibitors suggest an imbalance between increased proliferation, decreased apoptosis and increased ERK activity as a mechanism for WD defects in RetY1015F mice. Our work demonstrates novel inhibitory roles of RetY1015 and provides a possible mechanistic explanation for some of the confounding broad range phenotypes in individuals with CAKUT. PMID:22627285

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats.

PubMed

Melo, C H; Sousa, F C; Batista, R I P T; Sanchez, D J D; Souza-Fabjan, J M G; Freitas, V J F; Melo, L M; Teixeira, D I A

2015-07-31

The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.

Two FGFRL-Wnt circuits organize the planarian anteroposterior axis.

PubMed

Scimone, M Lucila; Cote, Lauren E; Rogers, Travis; Reddien, Peter W

2016-04-11

How positional information instructs adult tissue maintenance is poorly understood. Planarians undergo whole-body regeneration and tissue turnover, providing a model for adult positional information studies. Genes encoding secreted and transmembrane components of multiple developmental pathways are predominantly expressed in planarian muscle cells. Several of these genes regulate regional identity, consistent with muscle harboring positional information. Here, single-cell RNA-sequencing of 115 muscle cells from distinct anterior-posterior regions identified 44 regionally expressed genes, including multiple Wnt and ndk/FGF receptor-like (ndl/FGFRL) genes. Two distinct FGFRL-Wnt circuits, involving juxtaposed anterior FGFRL and posterior Wnt expression domains, controlled planarian head and trunk patterning. ndl-3 and wntP-2 inhibition expanded the trunk, forming ectopic mouths and secondary pharynges, which independently extended and ingested food. fz5/8-4 inhibition, like that of ndk and wntA, caused posterior brain expansion and ectopic eye formation. Our results suggest that FGFRL-Wnt circuits operate within a body-wide coordinate system to control adult axial positioning.

Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis.

PubMed

Ping, Suning; Liu, Shuying; Zhou, Yuhuan; Li, Ziqing; Li, Yuhuang; Liu, Kefeng; Bardeesi, Adham Sa; Wang, Linli; Chen, Jingbo; Deng, Lie; Wang, Jingjing; Wang, Hong; Chen, Dadi; Zhang, Zhengyu; Sheng, Puyi; Li, Chaohong

2017-05-25

Protein disulfide isomerase (PDI) involves cell survival and death. Whether PDI mediates mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs) -triggered simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is unknown. Here, we hypothesized that different expression levels of PDI trigger completely opposite cell fates among the different VSMC subtypes. Mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks; the grafted veins underwent simultaneous increases in proliferation and apoptosis, which triggered vein graft arterializations in non-diabetic or atherosclerosis in diabetic mice. A higher rate of proliferation and apoptosis was seen in the diabetic group. SS and/or AGEs stimulated the quiescent cultured VSMCs, resulting in simultaneous increases in proliferation and apoptosis; they could induce increased PDI activation and expression. Both in vivo and in vitro, the proliferating VSMCs indicated weak co-expression of PDI and SM-α-actin while apoptotic or dead cells showed strong co-expression of both. Either SS or AGEs rapidly upregulated the expression of PDI, NOX1 and ROS, and their combination had synergistic effects. Inhibiting PDI simultaneously suppressed the proliferation and apoptosis of VSMCs, while inhibition of SM-α-actin with cytochalasin D led to increased apoptosis and cleaved caspases-3 but had no effect on proliferation. In conclusion, different expression levels of PDI in VSMCs induced by SS and/or AGEs triggered a simultaneous increase in proliferation and apoptosis, accelerated vein graft arterializations or atherosclerosis, leading us to propose PDI as a novel target for the treatment of vascular remodeling and diseases.

Protein disulfide isomerase-mediated apoptosis and proliferation of vascular smooth muscle cells induced by mechanical stress and advanced glycosylation end products result in diabetic mouse vein graft atherosclerosis

PubMed Central

Ping, Suning; Liu, Shuying; Zhou, Yuhuan; Li, Ziqing; Li, Yuhuang; Liu, Kefeng; Bardeesi, Adham SA; Wang, Linli; Chen, Jingbo; Deng, Lie; Wang, Jingjing; Wang, Hong; Chen, Dadi; Zhang, Zhengyu; Sheng, Puyi; Li, Chaohong

2017-01-01

Protein disulfide isomerase (PDI) involves cell survival and death. Whether PDI mediates mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs) -triggered simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) is unknown. Here, we hypothesized that different expression levels of PDI trigger completely opposite cell fates among the different VSMC subtypes. Mouse veins were grafted into carotid arteries of non-diabetic and diabetic mice for 8 weeks; the grafted veins underwent simultaneous increases in proliferation and apoptosis, which triggered vein graft arterializations in non-diabetic or atherosclerosis in diabetic mice. A higher rate of proliferation and apoptosis was seen in the diabetic group. SS and/or AGEs stimulated the quiescent cultured VSMCs, resulting in simultaneous increases in proliferation and apoptosis; they could induce increased PDI activation and expression. Both in vivo and in vitro, the proliferating VSMCs indicated weak co-expression of PDI and SM-α-actin while apoptotic or dead cells showed strong co-expression of both. Either SS or AGEs rapidly upregulated the expression of PDI, NOX1 and ROS, and their combination had synergistic effects. Inhibiting PDI simultaneously suppressed the proliferation and apoptosis of VSMCs, while inhibition of SM-α-actin with cytochalasin D led to increased apoptosis and cleaved caspases-3 but had no effect on proliferation. In conclusion, different expression levels of PDI in VSMCs induced by SS and/or AGEs triggered a simultaneous increase in proliferation and apoptosis, accelerated vein graft arterializations or atherosclerosis, leading us to propose PDI as a novel target for the treatment of vascular remodeling and diseases. PMID:28542133

Interferon-Gamma and Fas Are Involved in Porphyromonas gingivalis-Induced Apoptosis of Human Extravillous Trophoblast-Derived HTR8/SVneo Cells via Extracellular Signal-Regulated Kinase 1/2 Pathway.

PubMed

Ren, Hongyu; Li, Yuhong; Jiang, Han; Du, Minquan

2016-11-01

A number of studies recently revealed a link between periodontal disease and preterm birth (PTB). PTB can be induced by dental infection with Porphyromonas gingivalis (Pg), a periodontopathic bacterium. This study aims to investigate responses of human extravillous trophoblast-derived HTR8/SVneo cells to Pg infection. Cell apoptosis, cell viability, protein expression, and cytokine production in HTR8 cells were measured via: 1) flow cytometry, 2) CCK-8 assay, 3) western blot, and 4) enzyme-linked immunosorbent assay methods, respectively. Pg decreased cell viability and increased cell apoptosis, active caspase-3 and Fas expression, and interferon-gamma (IFN-γ) secretion in HTR8 cells. Extracellular signal-regulated kinase (ERK) 1/2 inhibitor U0126 and FasL neutralizing antibody NOK1 that blocks FasL/Fas interaction both significantly suppressed Pg-induced apoptosis. U0126 also inhibited IFN-γ secretion and Fas expression close to control levels. Moreover, treatment with recombinant IFN-γ also significantly decreased number of viable HTR8 cells and increased Fas expression, suggesting IFN-γ may play an important role in Pg-induced apoptosis of HTR8 cells, at least partially through regulation of Fas expression. To the best of the authors' knowledge, this is the first study to demonstrate Pg induces IFN-γ secretion, Fas expression, and apoptosis in human extravillous trophoblast-derived HTR8/SVneo cells in an ERK1/2-dependent manner, and IFN-γ (explored by recombinant IFN-γ) and Fas are involved in Pg-induced apoptosis. The finding that Pg infection abnormally regulates inflammation and apoptosis of human trophoblasts may give new insights into the possible link of PTB with maternal periodontal disease and periodontal pathogens.

Bovine Herpes Virus 1 (BHV-1) and Herpes Simplex Virus Type 1 (HSV-1) Promote Survival of Latently Infected Sensory Neurons, in Part by Inhibiting Apoptosis

PubMed Central

Jones, Clinton

2013-01-01

α-Herpesvirinae subfamily members, including herpes simplex virus type 1 (HSV-1) and bovine herpes virus 1 (BHV-1), initiate infection in mucosal surfaces. BHV-1 and HSV-1 enter sensory neurons by cell-cell spread where a burst of viral gene expression occurs. When compared to non-neuronal cells, viral gene expression is quickly extinguished in sensory neurons resulting in neuronal survival and latency. The HSV-1 latency associated transcript (LAT), which is abundantly expressed in latently infected neurons, inhibits apoptosis, viral transcription, and productive infection, and directly or indirectly enhances reactivation from latency in small animal models. Three anti-apoptosis genes can be substituted for LAT, which will restore wild type levels of reactivation from latency to a LAT null mutant virus. Two small non-coding RNAs encoded by LAT possess anti-apoptosis functions in transfected cells. The BHV-1 latency related RNA (LR-RNA), like LAT, is abundantly expressed during latency. The LR-RNA encodes a protein (ORF2) and two microRNAs that are expressed in certain latently infected neurons. Wild-type expression of LR gene products is required for stress-induced reactivation from latency in cattle. ORF2 has anti-apoptosis functions and interacts with certain cellular transcription factors that stimulate viral transcription and productive infection. ORF2 is predicted to promote survival of infected neurons by inhibiting apoptosis and sequestering cellular transcription factors which stimulate productive infection. In addition, the LR encoded microRNAs inhibit viral transcription and apoptosis. In summary, the ability of BHV-1 and HSV-1 to interfere with apoptosis and productive infection in sensory neurons is crucial for the life-long latency-reactivation cycle in their respective hosts. PMID:25278776

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

PubMed

Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

2014-10-31

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Expression of natural killer cell activity with CD107a on ectopic endometrium in woman with endometriosis compared with non-endometriosis

NASA Astrophysics Data System (ADS)

Lubis, H. P.; Aldiansyah, D.; Siregar, H. S.; Rivany, R.; Hariadi, T. S.

2018-03-01

Some factors have an important role in endometriosis pathogenesis; there is an immune cell that plays an important role in endometrial cells that have reflux. Woman with endometriosis experienced the cellular immune disorder. It is suspected that decrease of NK cell in the peritoneal fluid caused by its qualitative defect with CD107a expression as the best marker. The aim of this study was to compare expression of NK Cell activity with CD107a between awoman with endometriosis and non-endometriosis. A case-control study from March until July 2015 in Haji Adam Malik General Hospital. The case group was ectopic endometrial tissue block paraffin and control group was normal endometrial tissue block paraffin. This study included 23 patients in endometriosis group and control group respectively. A majority proportion of CD107a expression in endometriosis group was +1 (16 patients (69.6%)), while the control group was +3 (9 patients (39.1%)). Expression of NK cell activity with CD107a in patients with endometriosis was lower than the control group (p<0.05). It suggested that cellular immune factors may play a role in the pathogenesis of endometriosis.

Interleukin-27 inhibits ectopic lymphoid-like structure development in early inflammatory arthritis

PubMed Central

Bombardieri, Michele; Greenhill, Claire J.; McLeod, Louise; Nerviani, Alessandra; Rocher-Ros, Vidalba; Cardus, Anna; Williams, Anwen S.; Pitzalis, Costantino; Jenkins, Brendan J.

2015-01-01

Ectopic lymphoid-like structures (ELSs) reminiscent of secondary lymphoid organs often develop at sites of chronic inflammation where they contribute to immune-mediated pathology. Through evaluation of synovial tissues from rheumatoid arthritis (RA) patients, we now show that low interleukin-27 (IL-27) expression corresponds with an increased incidence of ELS and gene signatures associated with their development and activity. The presence of synovial ELS was also noted in mice deficient in the IL-27 receptor (IL-27R) after the onset of inflammatory arthritis. Here, pathology was associated with increased synovial expression of pro-inflammatory cytokines, homeostatic chemokines, and transcriptional regulators linked with lymphoid neogenesis. In both clinical and experimental RA, synovial ELS coincided with the heightened local expression of cytokines and transcription factors of the Th17 and T follicular helper (Tfh) cell lineages, and included podoplanin-expressing T cells within lymphoid aggregates. IL-27 inhibited the differentiation of podoplanin-expressing Th17 cells, and an increased number of these cells were observed in IL-27R–deficient mice with inflammatory arthritis. Thus, IL-27 appears to negatively regulate ELS development in RA through control of effector T cells. These studies open new opportunities for patient stratification and treatment. PMID:26417004

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

PubMed Central

Nazer, Ezequiel; Dale, Ryan K.; Chinen, Madoka; Radmanesh, Behram

2018-01-01

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression. PMID:29529026

Induced stem cell neoplasia in a cnidarian by ectopic expression of a POU domain transcription factor.

PubMed

Millane, R Cathriona; Kanska, Justyna; Duffy, David J; Seoighe, Cathal; Cunningham, Stephen; Plickert, Günter; Frank, Uri

2011-06-01

The evolutionary origin of stem cell pluripotency is an unresolved question. In mammals, pluripotency is limited to early embryos and is induced and maintained by a small number of key transcription factors, of which the POU domain protein Oct4 is considered central. Clonal invertebrates, by contrast, possess pluripotent stem cells throughout their life, but the molecular mechanisms that control their pluripotency are poorly defined. To address this problem, we analyzed the expression pattern and function of Polynem (Pln), a POU domain gene from the marine cnidarian Hydractinia echinata. We show that Pln is expressed in the embryo and adult stem cells of the animal and that ectopic expression in epithelial cells induces stem cell neoplasms and loss of epithelial tissue. Neoplasm cells downregulated the transgene but expressed the endogenous Pln gene and also Nanos, Vasa, Piwi and Myc, which are all known cnidarian stem cell markers. Retinoic acid treatment caused downregulation of Pln and the differentiation of neoplasm cells to neurosensory and epithelial cells. Pln downregulation by RNAi led to differentiation. Collectively, our results suggest an ancient role of POU proteins as key regulators of animal stem cells.

Identification of Genes in the Phenylalanine Metabolic Pathway by Ectopic Expression of a MYB Transcription Factor in Tomato Fruit[W

PubMed Central

Dal Cin, Valeriano; Tieman, Denise M.; Tohge, Takayuki; McQuinn, Ryan; de Vos, Ric C.H.; Osorio, Sonia; Schmelz, Eric A.; Taylor, Mark G.; Smits-Kroon, Miriam T.; Schuurink, Robert C.; Haring, Michel A.; Giovannoni, James; Fernie, Alisdair R.; Klee, Harry J.

2011-01-01

Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and 13C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles. PMID:21750236

Translational repression by an RNA-binding protein promotes differentiation to infective forms in Trypanosoma cruzi.

PubMed

Romaniuk, Maria Albertina; Frasch, Alberto Carlos; Cassola, Alejandro

2018-06-01

Trypanosomes, protozoan parasites of medical importance, essentially rely on post-transcriptional mechanisms to regulate gene expression in insect vectors and vertebrate hosts. RNA binding proteins (RBPs) that associate to the 3'-UTR of mature mRNAs are thought to orchestrate master developmental programs for these processes to happen. Yet, the molecular mechanisms by which differentiation occurs remain largely unexplored in these human pathogens. Here, we show that ectopic inducible expression of the RBP TcUBP1 promotes the beginning of the differentiation process from non-infective epimastigotes to infective metacyclic trypomastigotes in Trypanosoma cruzi. In early-log epimastigotes TcUBP1 promoted a drop-like phenotype, which is characterized by the presence of metacyclogenesis hallmarks, namely repositioning of the kinetoplast, the expression of an infective-stage virulence factor such as trans-sialidase, increased resistance to lysis by human complement and growth arrest. Furthermore, TcUBP1-ectopic expression in non-infective late-log epimastigotes promoted full development into metacyclic trypomastigotes. TcUBP1-derived metacyclic trypomastigotes were infective in cultured cells, and developed normally into amastigotes in the cytoplasm. By artificial in vivo tethering of TcUBP1 to the 3' untranslated region of a reporter mRNA we were able to determine that translation of the reporter was reduced by 8-fold, while its mRNA abundance was not significantly compromised. Inducible ectopic expression of TcUBP1 confirmed its role as a translational repressor, revealing significant reduction in the translation rate of multiple proteins, a reduction of polysomes, and promoting the formation of mRNA granules. Expression of TcUBP1 truncated forms revealed the requirement of both N and C-terminal glutamine-rich low complexity sequences for the development of the drop-like phenotype in early-log epimastigotes. We propose that a rise in TcUBP1 levels, in synchrony with nutritional deficiency, can promote the differentiation of T. cruzi epimastigotes into infective metacyclic trypomastigotes.

Sessile serrated adenoma (SSA) vs. traditional serrated adenoma (TSA).

PubMed

Torlakovic, Emina Emilia; Gomez, Jose D; Driman, David K; Parfitt, Jeremy R; Wang, Chang; Benerjee, Tama; Snover, Dale C

2008-01-01

The morphologic distinction between various serrated polyps of the colorectum may be challenging. The distinction between sessile serrated adenoma (SSA) and traditional serrated adenoma (TSA) may be difficult using currently available criteria mostly based on cytologic characteristics. We have evaluated 66 serrated polyps including 29 SSA, 18 TSA, and 19 hyperplastic polyps for overall shape of the polyps, architectural features of individual crypts, the presence of eosinophilic cytoplasm, size and distribution of the proliferation and maturation zones, as well as Ki-67 and CK20 expression. The extent of the expression of CK20 and Ki-67 could not distinguish between the 3 types of serrated polyps, but the distribution of their expression was very helpful and differences were statistically significant. The distribution of Ki-67+ cells was the single most helpful distinguishing feature of the serrated polyp type (P<0.0001, chi test). Hyperplastic polyps had regular, symmetric, and increased Ki-67 expression. SSA had irregular, asymmetric, and highly variable expression of Ki-67. TSA had low Ki-67 expression, which was limited to "ectopic crypts" and admixed tubular adenomalike areas. In serrated polyps, ectopic crypt formation (ECF) defined by the presence of ectopic crypts with their bases not seated adjacent to the muscularis mucosae was nearly exclusive to TSA and was found in all cases, while the presence of cytologic atypia and eosinophilia of the cytoplasm were characteristic, but not limited to TSA. No evidence of ECF, but nevertheless abnormal distribution of proliferation zone was characteristic of SSA, whereas HP had neither. The presence of the ECF defines TSA in a more rigorous fashion than previous diagnostic criteria and also explains the biologic basis of exuberant protuberant growth associated with TSA and the lack of such growth in SSA. Recognition of this phenomenon may also help in exploring the genetic and molecular basis for differences between SSA and TSA, because these architectural abnormalities may well be a reflection of abnormalities in genetically programmed mucosal development.

Treatment-related neuroendocrine prostate cancer resulting in Cushing's syndrome.

PubMed

Ramalingam, Sundhar; Eisenberg, Adva; Foo, Wen Chi; Freedman, Jennifer; Armstrong, Andrew J; Moss, Larry G; Harrison, Michael R

2016-12-01

Here we present, to the best of our knowledge, the first case of a paraneoplastic Cushing's syndrome (hypercortisolism) resulting from treatment-related neuroendocrine prostate cancer - a highly aggressive and difficult disease to treat. A 51-year-old man was started on androgen deprivation therapy after presenting with metastatic prostate cancer, characterized by diffuse osseous metastasis. Shortly thereafter, he developed progressive disease with biopsy proven neuroendocrine prostate cancer as well as symptoms of increased skin pigmentation, hypokalemia, hypertension, hyperglycemia and profound weakness, consistent with ectopic Cushing's syndrome. Molecular analysis of the patient's tumor through RNA sequencing showed high expression of several genes including CHGA, ASCL1, CALCA, HES6, PCSK1, CALCB and INSM1 confirming his neuroendocrine phenotype; elevated POMC expression was found, supporting the diagnosis of ectopic Cushing's syndrome. © 2016 The Japanese Urological Association.

Bar represses dPax2 and decapentaplegic to regulate cell fate and morphogenetic cell death in Drosophila eye.

PubMed

Kang, Jongkyun; Yeom, Eunbyul; Lim, Janghoo; Choi, Kwang-Wook

2014-01-01

The coordinated regulation of cell fate and cell survival is crucial for normal pattern formation in developing organisms. In Drosophila compound eye development, crystalline arrays of hexagonal ommatidia are established by precise assembly of diverse cell types, including the photoreceptor cells, cone cells and interommatidial (IOM) pigment cells. The molecular basis for controlling the number of cone and IOM pigment cells during ommatidial pattern formation is not well understood. Here we present evidence that BarH1 and BarH2 homeobox genes are essential for eye patterning by inhibiting excess cone cell differentiation and promoting programmed death of IOM cells. Specifically, we show that loss of Bar from the undifferentiated retinal precursor cells leads to ectopic expression of Prospero and dPax2, two transcription factors essential for cone cell specification, resulting in excess cone cell differentiation. We also show that loss of Bar causes ectopic expression of the TGFβ homolog Decapentaplegic (Dpp) posterior to the morphogenetic furrow in the larval eye imaginal disc. The ectopic Dpp expression is not responsible for the formation of excess cone cells in Bar loss-of-function mutant eyes. Instead, it causes reduction in IOM cell death in the pupal stage by antagonizing the function of pro-apoptotic gene reaper. Taken together, this study suggests a novel regulatory mechanism in the control of developmental cell death in which the repression of Dpp by Bar in larval eye disc is essential for IOM cell death in pupal retina.

Venlafaxine inhibits apoptosis of hippocampal neurons by up-regulating brain-derived neurotrophic factor in a rat depression model

PubMed Central

Huang, Xiao; Mao, Yue-Shi; Li, Chao; Wang, Hao; Ji, Jian-Lin

2014-01-01

Objective: To study the effect of venlafaxine on the expression of brain-derived neurotrophic factor (BDNF) in rat hippocampal neurons, as well as its inhibitory effect on apoptosis of hippocampal neurons. Methods: Differences in behavioral ability between the depression model group and the Venlafaxine treatment group were observed using behavioral, sucrose-water and open field tests. The rat hippocampal tissue was sliced, stained and observed for BDNF distribution by immunohistochemistry. Apoptosis of hippocampal neurons was detected by TUNEL. BDNF expression in the hippocampal tissue was detected by Western blot. Injury and apoptosis of the hippocampal tissue were observed by electron microscopy. Results: Behavioral test showed that venlafaxine effectively improved the behavioral abilities of depressed rats. Immunohistochemistry showed that venlafaxine markedly increased the BDNF expression in the rat hippocampus. TUNEL showed that venlafaxine markedly inhibited apoptosis of hippocampal neurons, which was also confirmed by electron microscopic observation of the pathologic sections. Conclusion: Venlafaxine improved the expression of BDNF through working on PI3k/PKB/eNOS pathway and repressed the apoptosis of hippocampal neurons. PMID:25197330

Palmitate induces VSMC apoptosis via toll like receptor (TLR)4/ROS/p53 pathway.

PubMed

Zhang, Yuanjun; Xia, Guanghao; Zhang, Yaqiong; Liu, Juxiang; Liu, Xiaowei; Li, Weihua; Lv, Yaya; Wei, Suhong; Liu, Jing; Quan, Jinxing

2017-08-01

Toll-like receptor 4 (TLR4) has been implicated in vascular inflammation, as well as in the pathogenesis of atherosclerosis and diabetes. Vascular smooth muscle cell (VSMC) apoptosis has been shown to induce plaque vulnerability in atherosclerosis. Previous studies reported that palmitate induced apoptosis in VSMCs; however, the role of TLR4 in palmitate-induced apoptosis in VSMCs has not yet been defined. In this study, we investigated whether or not palmitate-induced apoptosis depended on the activation of the TLR4 pathway. VSMCs were treated with or without palmitate, CRISPR/Cas9z-mediated genome editing methods were used to deplete TLR4 expression, while NADPH oxidase inhibitors were used to inhibit reactive oxygen species (ROS) generation. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, ROS was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method, the mRNA and protein expression levels of caspase 3, caspase 9, BCL-2 and p53 were studied by real-time polymerase chain reaction (RT-PCR) and ELISA. Palmitate significantly promotes VSMC apoptosis, ROS generation, and expression of caspase 3, caspase 9 and p53; while NADPH oxidase inhibitor pretreatment markedly attenuated these effects. Moreover, knockdown of TLR4 significantly blocked palmitate-induced ROS generation and VSMC apoptosis accompanied by inhibition of caspase 3, caspase 9, p53 expression and restoration of BCL-2 expression. Our results suggest that palmitate-induced apoptosis depends on the activation of the TLR4/ROS/p53 signaling pathway, and that TLR4 may be a potential therapeutic target for the prevention and treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

AMP-activated Protein Kinase Mediates Apoptosis in Response to Bioenergetic Stress through Activation of the Pro-apoptotic Bcl-2 Homology Domain-3-only Protein BMF*

PubMed Central

Kilbride, Seán M.; Farrelly, Angela M.; Bonner, Caroline; Ward, Manus W.; Nyhan, Kristine C.; Concannon, Caoimhín G.; Wollheim, Claes B.; Byrne, Maria M.; Prehn, Jochen H. M.

2010-01-01

Heterozygous loss-of-function mutations in the hepatocyte nuclear factor 1A (HNF1A) gene result in the pathogenesis of maturity-onset diabetes-of-the-young type 3, (HNF1A-MODY). This disorder is characterized by a primary defect in metabolism-secretion coupling and decreased beta cell mass, attributed to excessive beta cell apoptosis. Here, we investigated the link between energy stress and apoptosis activation following HNF1A inactivation. This study employed single cell fluorescent microscopy, flow cytometry, gene expression analysis, and gene silencing to study the effects of overexpression of dominant-negative (DN)-HNF1A expression on cellular bioenergetics and apoptosis in INS-1 cells. Induction of DN-HNF1A expression led to reduced ATP levels and diminished the bioenergetic response to glucose. This was coupled with activation of the bioenergetic stress sensor AMP-activated protein kinase (AMPK), which preceded the onset of apoptosis. Pharmacological activation of AMPK using aminoimidazole carboxamide ribonucleotide (AICAR) was sufficient to induce apoptosis in naive cells. Conversely, inhibition of AMPK with compound C or AMPKα gene silencing protected against DN-HNF1A-induced apoptosis. Interestingly, AMPK mediated the induction of the pro-apoptotic Bcl-2 homology domain-3-only protein Bmf (Bcl-2-modifying factor). Bmf expression was also elevated in islets of DN-HNF1A transgenic mice. Furthermore, knockdown of Bmf expression in INS-1 cells using siRNA was sufficient to protect against DN-HNF1A-induced apoptosis. Our study suggests that overexpression of DN-HNF1A induces bioenergetic stress and activation of AMPK. This in turn mediates the transcriptional activation of the pro-apoptotic Bcl-2-homology protein BMF, coupling prolonged energy stress to apoptosis activation. PMID:20841353

Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

PubMed Central

Boos, Anja M; Loew, Johanna S; Deschler, Gloria; Arkudas, Andreas; Bleiziffer, Oliver; Gulle, Heinz; Dragu, Adrian; Kneser, Ulrich; Horch, Raymund E; Beier, Justus P

2011-01-01

Abstract Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto-transplanted versus in vitro expanded MSC with or without bone morphogenetic protein-2 (BMP-2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto-transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT-PCR analysis before subcutaneous implantation in combination with BMP-2 and β-tricalcium phosphate/hydroxyapatite (β-TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT-PCR evaluation. Sheep MSC were CD29+, CD44+ and CD166+ after selection by Ficoll gradient centrifugation, while directly auto-transplanted MSC-populations expressed CD29 and CD166 at lower levels. Both, directly auto-transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto-transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β-TCP/HA matrix comparable to the application of 60 μg/ml BMP-2 only or implantation of expanded MSC. Bone matrix proteins were up-regulated in constructs following direct auto-transplantation and in expanded MSC as well as in BMP-2 constructs. Up-regulation was detected using immunohistology methods and RT-PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto-transplanted or expanded MSC with β-TCP/HA granules alone. Hence BMP-2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering. PMID:20636333

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli.

PubMed

Dodi, Amos E; Ajayi, Iyabode O; Chang, Christine; Beard, Meghan; Ashley, Shanna L; Huang, Steven K; Thannickal, Victor J; Tschumperlin, Daniel J; Sisson, Thomas H; Horowitz, Jeffrey C

2018-05-10

Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

SPARC ectopic overexpression inhibits growth and promotes programmed cell death in acute myeloid leukemia transformed from myelodysplastic syndrome cells, alone and in combination with Ara-C treatment.

PubMed

Nian, Qing; Chi, Jianxiang; Xiao, Qing; Wei, Chunmei; Costeas, Paul; Yang, Zesong; Liu, Lin; Wang, Li

2015-09-01

Secreted protein acidic and rich in cysteine (SPARC) has a complex and pleiotropic biological role in cell life during disease. The role of SPARC in myelodysplastic syndrome (MDS) is not yet fully understood. In the present study, we investigated the role of SPARC protein overproduction in the proliferation and apoptosis of SKM-1 cells, an acute myeloid leukemia cell line transformed from MDS. SKM-1 cells were infected with the pGC-GV-SPARC vector. The cells were then assessed for proliferation and cell death following treatment with low-dose cytosine arabinoside (Ara‑C). The microarray analysis results revealed that samples from SPARC‑overexpressed cells compared to SPARC protein, in SKM-1 cells led to proliferation inhibition and promoted programmed cell death and these effects were greater when treated with Ara-C. The mRNA and protein expression levels of SPARC were detected by SPARC overexpression in cells treated with Ara-C resulting in a significant upregulation of the mixed lineage kinase domain-like (MLKL) gene expression and five other genes. The results showed that the necrotic signaling pathway may play a role when the two conditions were combined via the upregulation of the MLKL protein. MLKL upregulation in SPARC overexpressed cells treated with Ara-C, indicates necrosis as a possible cell death process for the SKM-1 cells under these stringent conditions.

BMPRIA Mediated Signaling Is Essential for Temporomandibular Joint Development in Mice

PubMed Central

Liu, Chao; Yang, Ling; Sun, Cheng; Ye, Wenduo; Li, Xihai; Chen, Jianquan; Long, Fanxin; Chen, YiPing

2014-01-01

The central importance of BMP signaling in the development and homeostasis of synovial joint of appendicular skeleton has been well documented, but its role in the development of temporomandibular joint (TMJ), also classified as a synovial joint, remains completely unknown. In this study, we investigated the function of BMPRIA mediated signaling in TMJ development in mice by transgenic loss-of- and gain-of-function approaches. We found that BMPRIA is expressed in the cranial neural crest (CNC)-derived developing condyle and glenoid fossa, major components of TMJ, as well as the interzone mesenchymal cells. Wnt1-Cre mediated tissue specific inactivation of BmprIa in CNC lineage led to defective TMJ development, including failure of articular disc separation from a hypoplastic condyle, persistence of interzone cells, and failed formation of a functional fibrocartilage layer on the articular surface of the glenoid fossa and condyle, which could be at least partially attributed to the down-regulation of Ihh in the developing condyle and inhibition of apoptosis in the interzone. On the other hand, augmented BMPRIA signaling by Wnt1-Cre driven expression of a constitutively active form of BmprIa (caBmprIa) inhibited osteogenesis of the glenoid fossa and converted the condylar primordium from secondary cartilage to primary cartilage associated with ectopic activation of Smad-dependent pathway but inhibition of JNK pathway, leading to TMJ agenesis. Our results present unambiguous evidence for an essential role of finely tuned BMPRIA mediated signaling in TMJ development. PMID:25093411

RSK2 is a new Pim2 target with pro-survival functions in FLT3-ITD-positive acute myeloid leukemia.

PubMed

Hospital, M-A; Jacquel, A; Mazed, F; Saland, E; Larrue, C; Mondesir, J; Birsen, R; Green, A S; Lambert, M; Sujobert, P; Gautier, E-F; Salnot, V; Le Gall, M; Decroocq, J; Poulain, L; Jacque, N; Fontenay, M; Kosmider, O; Récher, C; Auberger, P; Mayeux, P; Bouscary, D; Sarry, J-E; Tamburini, J

2018-03-01

Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.

Effects of naringin on the expression of miR-19b and cell apoptosis in human hepatocellular carcinoma

PubMed Central

Xie, Dafei; Yuan, Peiwen; Wang, Dong; Jin, Hua; Chen, Hui

2017-01-01

The effects of naringin on the expression of miR-19b and cell apoptosis were investigated in the human hepatocellular carcinoma cell line HepG2. HepG2 cells were treated with varied concentrations of naringin. The effects of naringin on the proliferation of HepG2 cells were observed by an MTT assay, morphological changes of cells were observed by an inverted microscope, cell apoptosis was detected by DAPI staining, miR-19b mRNA levels were determined with RT-PCR, and the expression of Bax and Bcl-2 proteins was examined by western blot assay. MTT results showed that naringin significantly inhibited the proliferation of HepG2 cells. Apoptotic HepG2 cells showed obvious changes in morphology under inverted microscope. DAPI staining suggested that naringin could induce cell shrinkage and nuclear chromatin condensation. RT-PCR results showed that naringin could upregulate the expression of miR-19b mRNA. Finally, western blot suggested that naringin upregulated the expression of Bax protein, but downregulated the expression of Bcl-2 protein. In conclusion, naringin can upregulate the expression of miR-19b mRNA and induce HepG2 cell apoptosis. In addition, it can also upregulate the expression of Bax protein and downregulate the expression of Bcl-2 protein during the process of apoptosis. PMID:28789364

Effects of naringin on the expression of miR-19b and cell apoptosis in human hepatocellular carcinoma.

PubMed

Xie, Dafei; Yuan, Peiwen; Wang, Dong; Jin, Hua; Chen, Hui

2017-08-01

The effects of naringin on the expression of miR-19b and cell apoptosis were investigated in the human hepatocellular carcinoma cell line HepG2. HepG2 cells were treated with varied concentrations of naringin. The effects of naringin on the proliferation of HepG2 cells were observed by an MTT assay, morphological changes of cells were observed by an inverted microscope, cell apoptosis was detected by DAPI staining, miR-19b mRNA levels were determined with RT-PCR, and the expression of Bax and Bcl-2 proteins was examined by western blot assay. MTT results showed that naringin significantly inhibited the proliferation of HepG2 cells. Apoptotic HepG2 cells showed obvious changes in morphology under inverted microscope. DAPI staining suggested that naringin could induce cell shrinkage and nuclear chromatin condensation. RT-PCR results showed that naringin could upregulate the expression of miR-19b mRNA. Finally, western blot suggested that naringin upregulated the expression of Bax protein, but downregulated the expression of Bcl-2 protein. In conclusion, naringin can upregulate the expression of miR-19b mRNA and induce HepG2 cell apoptosis. In addition, it can also upregulate the expression of Bax protein and downregulate the expression of Bcl-2 protein during the process of apoptosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Xiaohua; Xiao, Yipin; Chen, Chao, E-mail: chenchaopw@126.com

Elevated cytoplasmic polyadenylation element-binding 4 (CPEB4) is aberrantly expressed in several malignant cancers. However, its expression pattern, clinical significance, and biological function in colorectal cancer are still unknown. In this study, we demonstrated that CPEB4 is abundantly overexpressed in colorectal cancers and has the potential to be used for predicting clinical outcomes of colorectal cancer patients. We suppressed CPEB4 expression by small interfering RNA (siRNA) in SW480 and LOVO cells to clarify the role of CPEB4 on the cell apoptosis and proliferation in vitro. Further study revealed that knockdown of CPEB4 decreased the expression of anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL),more » but enhanced the expression of B-cell lymphoma-2-associated X (Bax). In addition, we indicated that CPEB4 is a novel target of miR-203, a tumor suppressive microRNA. Notably, restoration of CPEB4 in SW480 cells inhibited miR-203-induced apoptosis signaling pathway, which in turn enhanced cell proliferation and suppressed cell apoptosis. Taken together, our findings imply that posttranscriptional deregulation of CPEB4 contributes to the inhibited cell proliferation and the enhanced cell apoptosis in colorectal cancer, and directly targeting CPEB4 by miR-203 might be a novel strategy in colorectal cancer treatment. - Highlights: • CPEB4 is aberrantly expressed in human colorectal cancers. • Knockdown of CPEB4 inhibited colorectal cancer cell proliferation and enhanced apoptosis. • CPEB4 is a direct target of miR-203 and inversely correlates with miR-203 expression. • miR-203 inhibited cell growth and enhanced cell apoptosis in CPEB4 dependent manner. • miR-203 is an upstream regulator of the CPEB4-induced apoptosis pathway.« less

Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

PubMed

Hassen, Samar; Ali, Akhtar A; Kilaparty, Surya P; Al-Anbaky, Qudes A; Majeed, Waqar; Boman, Bruce M; Fields, Jeremy Z; Ali, Nawab

2016-01-01

The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an indicator of apoptosis, showed that MLH1 translocation only occurred in MMR proficient (SW480) cells upon induction of apoptosis further suggested a MSH2 dependent role of MLH1 in apoptosis. These data suggest a role of MLH1 in mediation of apoptosis in a MSH2-dependent manner. Taken together, our data supported an interdependence of mismatch repair proteins, particularly MLH1 and MSH2, in the mediation of apoptosis in human colorectal carcinoma cell lines.

Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro.

PubMed

Zhang, Xiao-Hui; Wu, Hong; Tang, Shu; Li, Qiao-Ning; Xu, Jiao; Zhang, Miao; Su, Ya-Nan; Yin, Bin; Zhao, Qi-Ling; Kemper, Nicole; Hartung, Joerg; Bao, En-Dong

2017-06-30

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken ( Gallus gallus ) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

[Roles of KLF5 in inhibition TNFα-induced SK-BR-3 breast cancer cell apoptosis].

PubMed

Shi, Jianhong; Liu, Caiyun; Zhang, Anyi; Cui, Naipeng; Wang, Bing; Chen, Baoping; Ma, Zhenfeng

2014-07-08

To explore the expression levels and roles of Krüpple-like factor 5 (KLF5) in tumor necrosis factor α (TNFα)-induced SK-BR-3 breast cancer cells. SK-BR-3 breast cancer cells were stimulated by TNFα at different concentrations (0, 1, 5, 10, 20 µg/L) for specified durations (0, 6, 12, 24, 36 h). Western blot was performed to detect KLF5 protein levels. Then Western blot and quantitative real-time PCR (qRT-PCR) were used to detect the expression levels of apoptosis genes. Flow cytometry and qRT-PCR were used to observe the effects of exogenous KLF5 on TNFα-induced apoptosis of SK-BR-3 breast cancer cell. KLF5 expression levels significantly decreased in TNFα-stimulated SK-BR-3 breast cancer cells in a concentration- and time-dependent manner. Quantitative RT-PCR results showed that TNFα up-regulate apoptosis gene caspase 3, caspase 9 and bax expression levels and down-regulate bcl-1 level in SK-BR-3 cells. Adenovirus expression vectors of pAd-GFP and pAd-GFP-KLF5 were constructed and used to infect SK-BR-3 breast cancer cells. Over-expression of GFP-KLF5 inhibited apoptosis in TNFα-stimulated SK-BR-3 breast cancer cells. TNFα reduces KLF5 expression in SK-BR-3 breast cancer cells and KLF5 participates in TNFα-induced SK-BR-3 cell apoptosis.

MicroRNA-21 regulates the proliferation and apoptosis of cervical cancer cells via tumor necrosis factor-α.

PubMed

Xu, Lin; Xu, Qian; Li, Xiwen; Zhang, Xiaoling

2017-10-01

The proliferation and apoptosis of tumor cells are regulated by a variety of microRNAs (miRs). miR‑21 can inhibit the apoptosis of cancer cells in vitro. Tumor necrosis factor α (TNF‑α) serves an important role in the induction of proliferation of cervical cancer cells. Previous studies have demonstrated that the expression level of miR‑21 is associated with TNF‑α expression in alveolar macrophages. However, to the best of our knowledge, whether miR‑21 regulates TNF‑α in cervical cells has not been reported. The present study was designed to investigate whether miR‑21 regulates TNF‑α expression, proliferation and apoptosis of cervical cancer cells. miR‑21, miR‑21 inhibitor and control miRNA were synthesized and transfected into HeLa cervical cancer cells. Reverse transcription‑quantitative polymerase chain reaction was used to measure the expression levels of miR‑21 and TNF‑α at the mRNA level. Western blotting was used to measure the expression levels of TNF‑α at the protein level. MTT assay and Hoechest‑33342 staining were used to measure the proliferation and apoptosis of HeLa cells. miR‑21 was identified to upregulate the mRNA and protein expression levels of TNF‑α. Furthermore, upregulation of TNF‑α enhanced the proliferation capability of HeLa cells. Changes in the expression levels of miR‑21 and TNF‑α did not significantly affect the apoptosis of Hela cells. In conclusion, the present study demonstrated that miR‑21 regulates the expression of TNF‑α in HeLa cells. Additionally, the expression level of TNF‑α was positively associated with the proliferation capability of Hela cells, but not apoptosis. Therefore, miR‑21 regulates the proliferation of HeLa cells through regulation of TNF‑α. These results provide novel potential therapeutic targets for the treatment of cervical cancer.

Analysis of the role of GADD153 in the control of apoptosis in NS0 myeloma cells.

PubMed

Lengwehasatit, Idsada; Dickson, Alan J

2002-12-30

Apoptosis can limit the maximum production of recombinant protein expression from cultured mammalian cells. This article focuses on the links between nutrient deprivation, ER perturbation, the regulation of (growth arrest and DNA damage inducible gene 153) GADD153 expression and apoptosis. During batch culture, decreases in glucose and glutamine correlated with an increase in apoptotic cells. This event was paralleled by a simultaneous increase in GADD153 expression. The expression of GADD153 in batch culture was suppressed by the addition of nutrients and with fed-batch culture the onset of apoptosis was delayed but not completely prevented. In defined stress conditions, glucose deprivation had the greatest effect on cell death when compared to glutamine deprivation or the addition of tunicamycin (an inhibitor of glycosylation), added to generate endoplasmic reticulum stress. However, the contribution of apoptosis to overall cell death (as judged by morphology) was smaller in conditions of glucose deprivation than in glutamine deprivation or tunicamycin treatment. Transient activation of GADD153 expression was found to occur in response to all stresses and occurred prior to detection of the onset of cell death. These results imply that GADD153 expression is either a trigger for apoptosis or offers a valid indicator of the likelihood of cell death arising from stresses of relevance to the bioreactor environment. Copyright 2002 Wiley Periodicals, Inc.

Activation of PPAR alpha by fenofibrate inhibits apoptosis in vascular adventitial fibroblasts partly through SIRT1-mediated deacetylation of FoxO1.

PubMed

Wang, Wei-Rong; Liu, En-Qi; Zhang, Ji-Ye; Li, Yan-Xiang; Yang, Xiao-Feng; He, Yan-Hao; Zhang, Wei; Jing, Ting; Lin, Rong

2015-10-15

Recent studies demonstrated that the ligand-activated transcription factor peroxisome proliferator-activated receptorα (PPARα) acts in association with histone deacetylase sirtuin 1 (SIRT1) in the regulation of metabolism and inflammation involved in cardiovascular diseases. PPARα activation also participates in the modulation of cell apoptosis. Our previous study found that SIRT1 inhibits the apoptosis of vascular adventitial fibroblasts (VAFs). However, whether the role of PPARα in apoptosis of VAFs is mediated by SIRT1 remains unknown. In this study, we aimed to determine the effect of PPARα agonist fenofibrate on cell apoptosis and SIRT1 expression and related mechanisms in ApoE(-/-) mice and VAFs in vitro. We found that fenofibrate inhibited cell apoptosis in vascular adventitia and up-regulated SIRT1 expression in aorta of ApoE(-/-) mice. Moreover, SIRT1 activator resveratrol (RSV) further enhanced these effects of fenofibrate. In vitro study showed that activation of PPARα by fenofibrate inhibited TNF-α-induced cell apoptosis and cell cycle arrest in VAFs. Meanwhile, fenofibrate up-regulated SIRT1 expression and inhibited SIRT1 translocation from nucleus to cytoplasm in VAFs stimulated with TNF-α. Moreover, the effects of fenofibrate on cell apoptosis and SIRT1 expression in VAFs were reversed by PPARα antagonist GW6471. Importantly, treatment of VAFs with SIRT1 siRNA or pcDNA3.1(+)-SIRT1 showed that the inhibitory effect of fenofibrate on cell apoptosis in VAFs through SIRT1. On the other hand, knockdown of FoxO1 decreased cell apoptosis of VAFs compared with fenofibrate group. Overexpression of FoxO1 increased cell apoptosis of VAFs compared with fenofibrate group. Further study found that fenofibrate decreased the expression of acetylated-FoxO1 in TNF-α-stimulated VAFs, which was abolished by SIRT1 knockdown. Taken together, these findings indicate that activation of PPARα by fenofibrate inhibits cell apoptosis in VAFs partly through the SIRT1-mediated deacetylation of FoxO1. Copyright © 2015 Elsevier Inc. All rights reserved.

6-Shogaol enhances renal carcinoma Caki cells to TRAIL-induced apoptosis through reactive oxygen species-mediated cytochrome c release and down-regulation of c-FLIP(L) expression.

PubMed

Han, Min Ae; Woo, Seon Min; Min, Kyoung-jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Kim, Sang Hyun; Choi, Yung Hyun; Kwon, Taeg Kyu

2015-02-25

6-Shogaol, a potent bioactive compound in ginger (Zingiber officinale Roscoe), has been reported for anti-inflammatory and anti-cancer activity. In this study, we investigated the effect of 6-shogaol to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. The combined treatment with 6-shogaol and TRAIL markedly induces apoptosis in various cancer cells (renal carcinoma Caki cells, breast carcinoma MDA-MB-231 cells and glioma U118MG cells), but not in normal mesangial cells and normal mouse kidney cells. 6-Shogaol reduced the mitochondrial membrane potential (MMP) and released cytochrome c from mitochondria to cytosol via Bax activation. Furthermore, we found that 6-shogaol induced down-regulation of c-FLIP(L) expression at the post-translational levels and the overexpression of c-FLIP(L) markedly inhibited 6-shogaol plus TRAIL-induced apoptosis. Moreover, 6-shogaol increased reactive oxygen species (ROS) production in Caki cells. Pretreatment with ROS scavengers attenuated 6-shogaol plus TRAIL-induced apoptosis through inhibition of MMP reduction and down-regulation of c-FLIP(L) expression. In addition, 6-gingerol, another phenolic alkanone isolated from ginger, did not enhance TRAIL-induced apoptosis and down-regulate c-FLIP(L) expression. Taken together, our results demonstrated that 6-shogaol enhances TRAIL-mediated apoptosis in renal carcinoma Caki cells via ROS-mediated cytochrome c release and down-regulation of c-FLIP(L) expression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Dynamic change of SGK expression and its role in neuron apoptosis after traumatic brain injury.

PubMed

Wu, Xinmin; Mao, Hui; Liu, Jiao; Xu, Jian; Cao, Jianhua; Gu, Xingxing; Cui, Gang

2013-01-01

Activation of specific signaling pathways in response to mechanical trauma causes delayed neuronal apoptosis; GSK-3β/β-catenin signaling plays a critical role in the apoptosis of neurons in CNS diseases, SGK was discovered as a regulator of GSK-3β/β-catenin pathway, The goal of this study was to determine if the mechanism of cell death or survival mediated by the SGK/GSK-3β/β-catenin pathway is involved in a rat model of TBI. Here, an acute traumatic brain injury model was applied to investigate the expression change and possible roles of SGK, Expression of SGK, and total-GSK-3β, phospho-GSK3β on ser-9, beta-catenin, and caspase-3 were examined by immunohistochemistry and Western blot analysis. Double immunofluorescent staining was used to observe the SGK localizations. Si-RNA was performed to identify whether SGK regulates neuron apoptosis via GSK-3β/β-catenin pathway, ultimately inhibit caspase-3 activation. Temporally, SGK expression showed an increase pattern after TBI and reached a peak at day 3. Spatially, SGK was widely expressed in the neuron, rarely in astrocytes and oligodendrocytes; in addition, the expression patterns of active caspase-3 and phospho-GSK3β were parallel with that of SGK, at the same time, the expression of β-catenin shows similarity with SGK. In vitro, to further investigate the function of SGK, a neuronal cell line PC12 was employed to establish an apoptosis model. We analyzed the association of SGK with apoptosis on PC12 cells by western blot, immunofluorescent labeling and siRNA. the results implied that SGK plays an important role in neuron apoptosis via the regulation of GSK3β/β-catenin signaling pathway; ultimately inhibit caspase-3 activation. Taken together, we inferred traumatic brain injury induced an upregulation of SGK in the central nervous system, which show a protective role in neuron apoptosis.

c-FLIP and the NOXA/Mcl-1 axis participate in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells.

PubMed

Zhao, Xiaofei; Kong, Feng; Wang, Lei; Zhang, Han

2017-01-01

Choroidal melanoma is the most common primary malignant intraocular tumor, and very few effective therapies are available to treat it. Our study aimed to understand whether pemetrexed plus cisplatin exerts a beneficial synergistic effect in human choroidal melanoma cells and to delineate the underlying molecular mechanism. To accomplish these aims, we treated choroidal melanoma cells with pemetrexed and cisplatin and assessed cell survival with SRB and MTT assays. Proteins were detected using western blotting analysis. NOXA and CHOP were knocked down with siRNA. We found that pemetrexed or cisplatin alone inhibited survival and induced apoptosis in human choroidal melanoma cells. Furthermore, the expression levels of c-FLIP, an anti-apoptotic protein in the extrinsic apoptosis pathway, and Mcl-1, an anti-apoptotic protein in the intrinsic apoptosis pathway, were decreased by pemetrexed or cisplatin respectively, while the expression of a pro-apoptotic protein in the intrinsic apoptosis pathway, NOXA, was up-regulated. Moreover, pemetrexed or cisplatin alone increased the protein expression of the endoplasmic reticulum stress markers IRE1α, Bip and CHOP. Silencing CHOP expression reduced NOXA expression. These findings suggest that the pemetrexed or cisplatin induced intrinsic apoptosis via activation of the ER stress response. Importantly, combining the two compounds more strongly induced apoptosis. Following the cotreatment, CHOP and NOXA expression increased, while c-FLIP and Mcl-1 expression decreased, and these effects were more pronounced than when using either compound alone. This result suggests that pemetrexed and cisplatin synergistically activate ER stress response-induced apoptosis in choroidal melanoma cells. To summarize, the c-FLIP and NOXA/Mcl-1 axis participated in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells. Intrinsic apoptosis was induced via activation of the ER stress response. Our study provides important mechanistic insights into potential cancer treatment with pemetrexed plus cisplatin and enriches our understanding of human choroidal melanoma.

p38 phosphorylation in medullary microglia mediates ectopic orofacial inflammatory pain in rats.

PubMed

Kiyomoto, Masaaki; Shinoda, Masamichi; Honda, Kuniya; Nakaya, Yuka; Dezawa, Ko; Katagiri, Ayano; Kamakura, Satoshi; Inoue, Tomio; Iwata, Koichi

2015-08-12

Orofacial inflammatory pain is likely to accompany referred pain in uninflamed orofacial structures. The ectopic pain precludes precise diagnosis and makes treatment problematic, because the underlying mechanism is not well understood. Using the established ectopic orofacial pain model induced by complete Freund's adjuvant (CFA) injection into trapezius muscle, we analyzed the possible role of p38 phosphorylation in activated microglia in ectopic orofacial pain. Mechanical allodynia in the lateral facial skin was induced following trapezius muscle inflammation, which accompanied microglial activation with p38 phosphorylation and hyperexcitability of wide dynamic range (WDR) neurons in the trigeminal spinal subnucleus caudalis (Vc). Intra-cisterna successive administration of a p38 mitogen-activated protein kinase selective inhibitor, SB203580, suppressed microglial activation and its phosphorylation of p38. Moreover, SB203580 administration completely suppressed mechanical allodynia in the lateral facial skin and enhanced WDR neuronal excitability in Vc. Microglial interleukin-1β over-expression in Vc was induced by trapezius muscle inflammation, which was significantly suppressed by SB203580 administration. These findings indicate that microglia, activated via p38 phosphorylation, play a pivotal role in WDR neuronal hyperexcitability, which accounts for the mechanical hypersensitivity in the lateral facial skin associated with trapezius muscle inflammation.

Apoptosis of T lymphocytes invading glioblastomas multiforme: a possible tumor defense mechanism.

PubMed

Didenko, Vladimir V; Ngo, Hop N; Minchew, Candace; Baskin, David S

2002-03-01

The goal of this study was to investigate whether apoptosis occurs in T lymphocytes that invade Fas ligand (FasL)-expressing glioblastomas multiforme (GBMs) and if its induction could be mediated by Fas. Apoptotic T lymphocytes were detected in GBMs by using detection of cell-type markers combined with active caspase-3 immunohistochemical analysis, a recently introduced apoptosis-specific in situ ligation assay, as well as by examining morphological criteria. Apoptotic T cells expressed Fas and were localized in the vicinity or in direct contact with FasL-expressing tumor cells. The T lymphocytes were undergoing apoptosis in spite of Bcl-2 expression. Expression of Bax was also detected in dying T cells, which can explain the absence of the protective effect of Bcl-2. because Bax inhibits Bcl-2 death-repressor activity. On the basis of the data presented in this paper, the authors suggest that GBM cells that express FasL can induce apoptosis in invading immune cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. Awareness of this phenomenon should be helpful for the development of novel strategies for treatment of malignant gliomas.

The remedial effect of soluble interleukin-1 receptor type II on endometriosis in the nude mouse model.

PubMed

Gao, Liying; Sun, Liang; Cui, Yugui; Hou, Zhen; Gao, Li; Zhou, Jing; Mao, Yundong; Han, Suping; Liu, Jiayin

2010-01-01

Recent studies have shown that the local expression of soluble interleukin (IL) -1 receptor type II (sIL-1 RII) in endometrial tissue of women with endometriosis is decreased, and the depression of IL-1 RII was more significant in infertile women than that in fertile women with endometriosis. In this research, we investigated the remedial effect of sIL-1-RII administration on endometriosis in the nude mouse model. NINETEEN NUDE MODEL MICE WITH ENDOMETRIOSIS WERE RANDOMLY DIVIDED INTO THREE GROUPS: group A was treated by intraperitoneal administration with only sIL-1 RII for two weeks, group B was similarly treated with only IL-1, and group C (control) was administered saline . After 2 weeks, the size of the ectopic endometrial lesions was calculated, and the expression of vascular endothelial growth factor (VEGF) and B-cell lymphoma leukemia-2 (Bcl-2) were detected by immunohistochemistry. The IL-8 and VEGF levels in the peritoneal fluid (PF) and serum were also measured by enzyme-linked immunosorbent assay (ELISA). The mean size of ectopic endometrial lesion did not differ between the three groups (P > 0.05). Compared with the control, the expression of VEGF and Bcl-2 was significantly lower in group A, and higher in group B. In the three groups, the levels of IL-8 in the PF and serum were highest in group A, and lowest in group B. sIL-1 RII may suppresse hyperplasia of ectopic endometriosis, perhaps by reducing the expression of certain cytokines, such as VEGF, IL-8, and Bcl-2, which could provide a new clinical strategy for the treatment of endometriosis.

Small Heat Shock Proteins Can Release Light Dependence of Tobacco Seed during Germination1[OPEN

PubMed Central

Koo, Hyun Jo; Park, Soo Min; Kim, Keun Pill; Suh, Mi Chung; Lee, Mi Ok; Lee, Seong-Kon; Xinli, Xia

2015-01-01

Small heat shock proteins (sHSPs) function as ATP-independent molecular chaperones, and although the production and function of sHSPs have often been described under heat stress, the expression and function of sHSPs in fundamental developmental processes, such as pollen and seed development, have also been confirmed. Seed germination involves the breaking of dormancy and the resumption of embryo growth that accompany global changes in transcription, translation, and metabolism. In many plants, germination is triggered simply by imbibition of water; however, different seeds require different conditions in addition to water. For small-seeded plants, like Arabidopsis (Arabidopsis thaliana), lettuce (Lactuca sativa), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum), light is an important regulator of seed germination. The facts that sHSPs accumulate during seed development, sHSPs interact with various client proteins, and seed germination accompanies synthesis and/or activation of diverse proteins led us to investigate the role of sHSPs in seed germination, especially in the context of light dependence. In this study, we have built transgenic tobacco plants that ectopically express sHSP, and the effect was germination of the seeds in the dark. Administering heat shock to the seeds also resulted in the alleviation of light dependence during seed germination. Subcellular localization of ectopically expressed sHSP was mainly observed in the cytoplasm, whereas heat shock-induced sHSPs were transported to the nucleus. We hypothesize that ectopically expressed sHSPs in the cytoplasm led the status of cytoplasmic proteins involved in seed germination to function during germination without additional stimulus and that heat shock can be another signal that induces seed germination. PMID:25604531

Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates Ectopic FoxE3 Expression and Disruption in Fiber Cell Differentiation

PubMed Central

Kerr, Christine L.; Huang, Jian; Williams, Trevor; West-Mays, Judith A.

2012-01-01

Purpose. The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. Methods. We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. Results. Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. Conclusions. Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3. PMID:22491411

Modulation of iridovirus-induced apoptosis by endocytosis, early expression, JNK, and apical caspase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chitnis, Nilesh S.; D'Costa, Susan M.; Paul, Eric R.

Chilo iridescent virus (CIV) is the type species for the family Iridoviridae, which are large, isometric, cytoplasmic dsDNA viruses. We examined the mechanism of apoptosis induction by CIV. High CIV doses (CIV{sub XS}; 400 {mu}g/ml), UV-irradiated virus (CIV{sub UV}; 10 {mu}g/ml) and CVPE (CIV protein extract; 10 {mu}g/ml) induced apoptosis in 60% of treated Choristoneura fumiferana (IPRI-CF-124T) cells. Normal doses of infectious CIV (10 {mu}g/ml) induced apoptosis in only 10% of C. fumiferana (CF) cells. Apoptosis was inhibited by Z-IETD-FMK, an apical caspase inhibitor, indicating that CIV-induced apoptosis requires caspase activity. The putative caspase in CF cells was designated Cf-caspase-i.more » CIV{sub UV} or CVPE enhanced Cf-caspase-i activity by 80% at 24 h relative to mock-treated cells. Since the MAP kinase pathway induces or inhibits apoptosis depending on the context, we used JNK inhibitor SP600125 and demonstrated drastic suppression of CVPE-induced apoptosis. Thus, the JNK signaling pathway is significant for apoptosis in this system. Virus interaction with the cell surface was not sufficient for apoptosis since CIV{sub UV} particles bound to polysterene beads failed to induce apoptosis. Endocytosis inhibitors (bafilomycin or ammonium chloride) negated apoptosis induction by CIV{sub UV}, CIV{sub XS} or CVPE indicating that entry through this mode is required. Given the weak apoptotic response to infectious CIV, we postulated that viral gene expression inhibited apoptosis. CIV infection of cells pretreated with cycloheximide induced apoptosis in 69% of the cells compared to 10% in normal infections. Furthermore, blocking viral DNA replication with aphidicolin or phosphonoacetic acid suppressed apoptosis and Cf-caspase-i activity, indicating that early viral expression is necessary for inhibition of apoptosis, and de novo synthesis of viral proteins is not required for induction. We show for the first time that, in a member of the family Iridoviridae, apoptosis: (i) requires entry and endocytosis of virions or virion proteins, (ii) is inhibited under conditions permitting early viral expression, and (iii) requires the JNK signaling pathway. This is the first report of JNK signal requirement during apoptosis induction by an insect virus.« less

Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer.

PubMed

Davila, Monica; Jhala, Darshana; Ghosh, Debashis; Grizzle, William E; Chakrabarti, Ratna

2007-06-08

LIM kinase 1 (LIMK1), a LIM domain containing serine/threonine kinase, modulates actin dynamics through inactivation of the actin depolymerizing protein cofilin. Recent studies have indicated an important role of LIMK1 in growth and invasion of prostate and breast cancer cells; however, the molecular mechanism whereby LIMK1 induces tumor progression is unknown. In this study, we investigated the effects of ectopic expression of LIMK1 on cellular morphology, cell cycle progression and expression profile of LIMK1 in prostate tumors. Ectopic expression of LIMK1 in benign prostatic hyperplasia cells (BPH), which naturally express low levels of LIMK1, resulted in appearance of abnormal mitotic spindles, multiple centrosomes and smaller chromosomal masses. Furthermore, a transient G1/S phase arrest and delayed G2/M progression was observed in BPH cells expressing LIMK1. When treated with chemotherapeutic agent Taxol, no metaphase arrest was noted in these cells. We have also noted increased nuclear staining of LIMK1 in tumors with higher Gleason Scores and incidence of metastasis. Our results show that increased expression of LIMK1 results in chromosomal abnormalities, aberrant cell cycle progression and alteration of normal cellular response to microtubule stabilizing agent Taxol; and that LIMK1 expression may be associated with cancerous phenotype of the prostate.

Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells.

PubMed

Shen, Miaoqing; Bunaciu, Rodica P; Congleton, Johanna; Jensen, Holly A; Sayam, Lavanya G; Varner, Jeffrey D; Yen, Andrew

2011-12-01

All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

Junction region of EWS-FLI1 fusion protein has a dominant negative effect in Ewing's sarcoma in vitro.

PubMed

Jully, Babu; Vijayalakshmi, Ramshankar; Gopal, Gopisetty; Sabitha, Kesavan; Rajkumar, Thangarajan

2012-11-12

Ewing's sarcoma is a malignancy characterized by a specific 11:22 chromosomal translocation which generates a novel EWS-FLI1 fusion protein functioning as an aberrant transcription factor. In the present study, we have further characterized the junction region of the EWS-FLI1 fusion protein. In-silico model of EWS-FLI1 fusion protein was analysed for ligand binding sites, and a putative region (amino acid (aa) 251-343 of the type 1 fusion protein) in the vicinity of the fusion junction was cloned and expressed using bacterial expression. The recombinant protein was characterized by Circular Dichroism (CD). We then expressed aa 251-280 ectopically in Ewing's sarcoma cell-line and its effect on cell proliferation, tumorigenicity and expression of EWS-FLI1 target genes were analysed. Our modelling analysis indicated that Junction region (aa 251-343) encompasses potential ligand biding sites in the EWS-FLI1 protein and when expressed in bacteria was present as soluble form. Ectopically expressing this region in Ewing's sarcoma cells inhibited tumorigenicity, and EWS-FLI1 target genes indicating a dominant negative biological effect. Junction region can be exploited further as target for drug development in future to specifically target EWS-FLI1 in Ewing's Sarcoma.

Ectopic expression of SUPERMAN suppresses development of petals and stamens.

PubMed

Yun, Jae-Young; Weigel, Detlef; Lee, Ilha

2002-01-01

The floral regulatory gene SUPERMAN (SUP) encodes a C2H2 type zinc finger protein that is required for maintaining boundaries between floral organs in Arabidopsis. It has been proposed that the main function of SUP is to balance cell proliferation in the third and fourth whorl of developing flowers, thereby maintaining the boundaries between the two whorls. To gain further insight into the function of SUP, we have ectopically expressed SUP using the promoter of APETALA1 (AP1), a gene that is initially expressed throughout floral meristems and later becomes restricted to the first and second whorls. Flowers of AP1::SUP plants have fewer floral organs, consistent with an effect of SUP on cell proliferation. In addition, the AP1::SUP transgene caused the conversion of petals to sepals and suppressed the development of stamens. The expression of the B function homeotic gene APETALA3 (AP3) and its regulator UNUSUAL FLORAL ORGANS (UFO) were delayed and reduced in AP1::SUP flowers. However, SUP does not act merely through UFO, as constitutive expression of UFO did not rescue the defects in petal and stamen development in AP1::SUP flowers. Together, these results suggest that SUP has both indirect and direct effects on the expression of B function homeotic genes.

Neuropilin-1 Is Expressed on Lymphoid Tissue Residing LTi-like Group 3 Innate Lymphoid Cells and Associated with Ectopic Lymphoid Aggregates.

PubMed

Shikhagaie, Medya Mara; Björklund, Åsa K; Mjösberg, Jenny; Erjefält, Jonas S; Cornelissen, Anne S; Ros, Xavier Romero; Bal, Suzanne M; Koning, Jasper J; Mebius, Reina E; Mori, Michiko; Bruchard, Melanie; Blom, Bianca; Spits, Hergen

2017-02-14

Here, we characterize a subset of ILC3s that express Neuropilin1 (NRP1) and are present in lymphoid tissues, but not in the peripheral blood or skin. NRP1 + group 3 innate lymphoid cells (ILC3s) display in vitro lymphoid tissue inducer (LTi) activity. In agreement with this, NRP1 + ILC3s are mainly located in proximity to high endothelial venules (HEVs) and express cell surface molecules involved in lymphocyte migration in secondary lymphoid tissues via HEVs. NRP1 was also expressed on mouse fetal LTi cells, indicating that NRP1 is a conserved marker for LTi cells. Human NRP1 + ILC3s are primed cells because they express CD45RO and produce higher amounts of cytokines than NRP1 - cells, which express CD45RA. The NRP1 ligand vascular endothelial growth factor A (VEGF-A) served as a chemotactic factor for NRP1 + ILC3s. NRP1 + ILC3s are present in lung tissues from smokers and patients with chronic obstructive pulmonary disease, suggesting a role in angiogenesis and/or the initiation of ectopic pulmonary lymphoid aggregates. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Ectopic expression of Arabidopsis Target of Rapamycin (AtTOR) improves water-use efficiency and yield potential in rice

NASA Astrophysics Data System (ADS)

Bakshi, Achala; Moin, Mazahar; Kumar, M. Udaya; Reddy, Aramati Bindu Madhava; Ren, Maozhi; Datla, Raju; Siddiq, E. A.; Kirti, P. B.

2017-02-01

The target of Rapamycin (TOR) present in all eukaryotes is a multifunctional protein, regulating growth, development, protein translation, ribosome biogenesis, nutrient, and energy signaling. In the present study, ectopic expression of TOR gene of Arabidopsis thaliana in a widely cultivated indica rice resulted in enhanced plant growth under water-limiting conditions conferring agronomically important water-use efficiency (WUE) trait. The AtTOR high expression lines of rice exhibited profuse tillering, increased panicle length, increased plant height, high photosynthetic efficiency, chlorophyll content and low Δ13C. Δ13C, which is inversely related to high WUE, was as low as 17‰ in two AtTOR high expression lines. These lines were also insensitive to the ABA-mediated inhibition of seed germination. The significant upregulation of 15 stress-specific genes in high expression lines indicates their contribution to abiotic stress tolerance. The constitutive expression of AtTOR is also associated with significant transcriptional upregulation of putative TOR complex-1 components, OsRaptor and OsLST8. Glucose-mediated transcriptional activation of AtTOR gene enhanced lateral root formation. Taken together, our findings indicate that TOR, in addition to its multiple cellular functions, also plays an important role in response to abiotic stress and potentially enhances WUE and yield related attributes.

High-Fat Diet-Induced Adiposity, Adipose Inflammation, Hepatic Steatosis and Hyperinsulinemia in Outbred CD-1 Mice

PubMed Central

Gao, Mingming; Ma, Yongjie; Liu, Dexi

2015-01-01

High-fat diet (HFD) has been applied to a variety of inbred mouse strains to induce obesity and obesity related metabolic complications. In this study, we determined HFD induced development of metabolic disorders on outbred female CD-1 mice in a time dependent manner. Compared to mice on regular chow, HFD-fed CD-1 mice gradually gained more fat mass and consequently exhibited accelerated body weight gain, which was associated with adipocyte hypertrophy and up-regulated expression of adipose inflammatory chemokines and cytokines such as Mcp-1 and Tnf-α. Increased fat accumulation in white adipose tissue subsequently led to ectopic fat deposition in brown adipose tissue, giving rise to whitening of brown adipose tissue without altering plasma level of triglyceride. Ectopic fat deposition was also observed in the liver, which was associated with elevated expression of key genes involved in hepatic lipid sequestration, including Ppar-γ2, Cd36 and Mgat1. Notably, adipose chronic inflammation and ectopic lipid deposition in the liver and brown fat were accompanied by glucose intolerance and insulin resistance, which was correlated with hyperinsulinemia and pancreatic islet hypertrophy. Collectively, these results demonstrate sequentially the events that HFD induces physiological changes leading to metabolic disorders in an outbred mouse model more closely resembling heterogeneity of the human population. PMID:25768847

High-fat diet-induced adiposity, adipose inflammation, hepatic steatosis and hyperinsulinemia in outbred CD-1 mice.

PubMed

Gao, Mingming; Ma, Yongjie; Liu, Dexi

2015-01-01

High-fat diet (HFD) has been applied to a variety of inbred mouse strains to induce obesity and obesity related metabolic complications. In this study, we determined HFD induced development of metabolic disorders on outbred female CD-1 mice in a time dependent manner. Compared to mice on regular chow, HFD-fed CD-1 mice gradually gained more fat mass and consequently exhibited accelerated body weight gain, which was associated with adipocyte hypertrophy and up-regulated expression of adipose inflammatory chemokines and cytokines such as Mcp-1 and Tnf-α. Increased fat accumulation in white adipose tissue subsequently led to ectopic fat deposition in brown adipose tissue, giving rise to whitening of brown adipose tissue without altering plasma level of triglyceride. Ectopic fat deposition was also observed in the liver, which was associated with elevated expression of key genes involved in hepatic lipid sequestration, including Ppar-γ2, Cd36 and Mgat1. Notably, adipose chronic inflammation and ectopic lipid deposition in the liver and brown fat were accompanied by glucose intolerance and insulin resistance, which was correlated with hyperinsulinemia and pancreatic islet hypertrophy. Collectively, these results demonstrate sequentially the events that HFD induces physiological changes leading to metabolic disorders in an outbred mouse model more closely resembling heterogeneity of the human population.

Expression of a monothiol glutaredoxin, AtGRXS17, in tomato (Solanum lycopersicum) enhances drought tolerance

USDA-ARS?s Scientific Manuscript database

Abiotic stresses are a major factor limiting crop growth and productivity. Our previous studies revealed that Arabidopsis thaliana glutaredoxin S17 (AtGRXS17) has conserved functions in plant tolerance to heat and chilling stress in tomato. Here, we report that ectopic expression of AtGRXS17 in toma...

VCC-1 over-expression inhibits cisplatin-induced apoptosis in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Zhitao; Lu, Xiao; Zhu, Ping

Highlights: Black-Right-Pointing-Pointer VCC-1 is hypothesized to be associated with carcinogenesis. Black-Right-Pointing-Pointer Levels of VCC-1 are increased significantly in HCC. Black-Right-Pointing-Pointer Over-expression of VCC-1 could promotes cellular proliferation rate. Black-Right-Pointing-Pointer Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. Black-Right-Pointing-Pointer VCC-1 plays an important role in control the tumor growth and apoptosis. -- Abstract: Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellularmore » carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy.« less

Preconditioning With Tauroursodeoxycholic Acid Protects Against Contrast-Induced HK-2 Cell Apoptosis by Inhibiting Endoplasmic Reticulum Stress.

PubMed

Peng, Pingan; Ma, Qian; Wang, Le; Zhang, Ou; Han, Hongya; Liu, Xiaoli; Zhou, Yujie; Zhao, Yingxin

2015-11-01

To investigate whether tauroursodeoxycholic acid (TUDCA) could attenuate contrast media (CM)-induced renal tubular cell apoptosis by inhibiting endoplasmic reticulum stress (ERS), we exposed HK-2 cells to increasing doses of meglumine diatrizoate (20, 40, and 80 mg I/mL) for 2 to 16 hours, with/without TUDCA preconditioning for 24 hours. Cell viability test, Hoechst 33258 staining, and flow cytometry were used to detect meglumine diatrizoate-induced cell apoptosis, while real-time polymerase chain reaction and Western blot analysis were used to measure the expressions of ERS markers of glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), and the apoptosis-related marker of caspase 12. Cell apoptosis and messenger RNA (mRNA) expression of GRP78 (P = .005), ATF4 (P = .01), and caspase 12 (P = .001) were significantly higher in the CM 4 hours group than the control as well as the protein expressions. The TUDCA preconditioning reduced the mRNA expression of GRP78, ATF4, and caspase 12 in the CM 4 hours groups (P = .009, .019, and .003, respectively) as well as the protein expression. In conclusion, TUDCA could protect renal tubular cells from meglumine diatrizoate-induced apoptosis by inhibiting ERS. © The Author(s) 2015.

Dacarbazine inhibits proliferation of melanoma FEMX-1 cells by up-regulating expression of miRNA-200.

PubMed

Chen, Y-N

2017-03-01

Melanoma is a highly aggressive tumour, and treatment efficacy depends on the stage of the tumour. Early stage cutaneous melanoma is efficiently treated by surgical excision. In contrast, late-stage melanoma requires chemotherapy with dacarbazine (DTIC). Unfortunately, advanced melanoma can often be resistant to DTIC. The mechanisms of anti-melanoma effects of DTIC are still poorly understood, which hinders development of more potent therapies. In this study, we examined the effects of DTIC on growth inhibition of FEMX-1 melanoma cell line, expression of apoptosis-related proteins, and expression of micro (mi)RNA-200 (miRNA-200a, miRNA-200b, miRNA-200c, and miRNA-141). DTIC was used at 50 (low dose) or 100 (high dose) mg/ml. Cell growth inhibition was documented by MTT assay. Cell apoptosis was quantified by propidium iodide staining and caspase 3-8 activity assay. Expression of apoptosis-related proteins Bim, Bak, BAX, and Bad were documented by Western blot analysis, while expression of miRNA-200 by PCR. DTIC dose-dependently inhibited growth of FEMX-1 melanoma cell line, induced cell apoptosis, modulated the levels of apoptosis-related proteins, and up-regulated expression of miRNA-200 family members. DTIC inhibits the growth of melanoma cells by up-regulating expression of miRNA-200.

Osthole inhibits proliferation and induces apoptosis in human osteosarcoma cells.

PubMed

Ding, Yong; Lu, Xiongwei; Hu, Xiaopeng; Ma, Jie; Ding, Huan

2014-02-01

The purpose of this study was to investigate the effect of osthole on osteosarcoma cell proliferation and apoptosis. Cell counting Kit-8 assay was performed to establish the effects of osthole on osteosarcoma MG-63 cell proliferation. Annexin V-FITC/PI was performed to analyze the apoptotic rate of the cells. The inhibitory effects of osthole on the expression of BCL-2, BAX, and caspase-3 were detected by Western blotting. Osthole inhibited the growth of human osteosarcoma MG-63 cells by inhibiting cell proliferation and induced cell apoptosis. Western blotting demonstrated that osthole downregulated the expressions of BCL-2 and caspase-3 and upregulated the expression of BAX in human osteosarcoma cells. Osthole can inhibit osteosarcoma cell proliferation and induced apoptosis effectively in a dose-dependent manner through downregulating the expression of BCL-2 and caspase-3 proteins levels and upregulating the expression of BAX proteins levels.

Fuzheng Huayu recipe alleviates hepatic fibrosis via inhibiting TNF-α induced hepatocyte apoptosis.

PubMed

Tao, Yan-yan; Yan, Xiu-chuan; Zhou, Tao; Shen, Li; Liu, Zu-long; Liu, Cheng-hai

2014-11-18

What was the relationship of Fuzheng Huayu recipe (FZHY) inhibiting hepatocyte apoptosis and HSC activation at different stage of liver fibrosis? In order to answer this question, the study was carried out to dynamically observe FZHY's effect on hepatocyte apoptosis and HSC activation and further explored underling mechanism of FZHY against hepatocyte apoptosis. Mice were randomly divided into four groups: normal, model, FZHY, and N-acetylcystein (NAC) groups. Acute hepatic injury and liver fibrosis in mice were induced by CCl4. Three days before the first CCl4 injection, treatment with FZHY powder or NAC respectively was started. In vitro, primary hepatocytes were pretreated with FZHY medicated serum or Z-VAD-FMK and then incubated with ActD and TNF-α. Primary HSCs were treated with DNA from apoptotic hepatocytes incubated by Act D/TNF-α or FZHY medicated. Liver sections were analyzed for HE staining and immunohistochemical evaluation of apoptosis. Serum ALT and AST, Alb content and TNF-α expression in liver tissue were detected. Hyp content was assayed and collagen deposition was visualized. Expressions of α-SMA and type I collagen were analyzed by immunofluorescence and immunoblotting. Flow cytometry, immunofluorescence, and DNA ladder for hepatocyte apoptosis and immunoblotting for TNF-R1, Bcl-2 and Bax were also analyzed. Mice showed characteristic features of massive hepatocytes apoptosis in early stage of liver injury and developed severe hepatic fibrosis in later phase. FZHY treatment significantly alleviated acute liver injury and hepatocyte apoptosis, and inhibited liver fibrosis by decreasing α-SMA expression and hepatic Hyp content. In vitro, primary hepatocytes were induced by TNF-α and Act D. The anti-apoptotic effect of FZHY was generated by reducing TNFR1 expression and balancing the expressions of Bcl-2 and Bax. Meanwhile, the nuclear DNA from apoptotic hepatocytes stimulated HSC activation in a dose dependent manner, and the DNA from apoptotic hepatocytes treated with FZHY or Z-VAD-FMK reduced HSC activation and type I collagen expression. These findings suggested that FZHY suppressed hepatocyte apoptosis through regulating mediators in death receptor and mitochondrial pathways, and the effect of FZHY on hepatocyte apoptosis might play an important role in inhibiting liver fibrosis.

Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway

PubMed Central

Wang, Xiuwen; Li, Ji; Wu, Dongjin; Bu, Xiangpeng

2015-01-01

Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia–reperfusion injury. PMID:26350953

HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP).

PubMed

Krueger, Andreas; Fas, Stefanie C; Giaisi, Marco; Bleumink, Marc; Merling, Anette; Stumpf, Christine; Baumann, Sven; Holtkotte, Denise; Bosch, Valerie; Krammer, Peter H; Li-Weber, Min

2006-05-15

The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax-expressing T-cell lines derived from HTLV-1-infected patients express elevated levels of c-FLIP(L) and c-FLIP(S). The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.

Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

PubMed Central

Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

2017-01-01

Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer. PMID:28382282

MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis.

PubMed

Chen, Q; Xu, J; Li, L; Li, H; Mao, S; Zhang, F; Zen, K; Zhang, C-Y; Zhang, Q

2014-03-20

Expression of apoptotic protease activating factor-1 (Apaf-1) gradually decreases during brain development, and this decrease is likely responsible for the decreased sensitivity of brain tissue to apoptosis. However, the mechanism by which Apaf-1 expression is decreased remains elusive. In the present study, we found that four microRNAs (miR-23a/b and miR-27a/b) of miR-23a-27a-24 and miR-23b-27b-24 clusters play key roles in modulating the expression of Apaf-1. First, we found that miR-23a/b and miR-27a/b suppressed the expression of Apaf-1 in vitro. Interestingly, the expression of the miR-23-27-24 clusters in the mouse cortex gradually increased in a manner that was inversely correlated with the pattern of Apaf-1 expression. Second, hypoxic injuries during fetal distress caused reduced expression of the miR-23b and miR-27b that was inversely correlated with an elevation of Apaf-1 expression during neuronal apoptosis. Third, we made neuronal-specific transgenic mice and found that overexpressing the miR-23b and miR-27b in mouse neurons inhibited the neuronal apoptosis induced by intrauterine hypoxia. In conclusion, our results demonstrate, in central neural system, that miR-23a/b and miR-27a/b are endogenous inhibitory factors of Apaf-1 expression and regulate the sensitivity of neurons to apoptosis. Our findings may also have implications for the potential target role of microRNAs in the treatment of neuronal apoptosis-related diseases.

Effects of hypo- and hyperthyroidism on proliferation, angiogenesis, apoptosis and expression of COX-2 in the corpus luteum of female rats.

PubMed

Silva, J F; Ocarino, N M; Vieira, A L S; Nascimento, E F; Serakides, R

2013-08-01

Although thyroid dysfunction occurs frequently in humans and some animal species, the mechanisms by which hypo- and hyperthyroidism affect the corpus luteum have not been thoroughly elucidated. This study evaluated the levels of proliferative activity, angiogenesis, apoptosis and expression of cyclooxygenase-2 in the corpus luteum of female rats with thyroid dysfunction. These processes may be important in understanding the reproductive changes caused by thyroid dysfunction. A total of 18 adult female rats were divided into three groups (control, hypothyroid and hyperthyroid) with six animals per group. Three months after treatment to induce thyroid dysfunction, the rats were euthanized in the dioestrus phase. The ovaries were collected and immunohistochemically analysed for expression of the cell proliferation marker CDC-47, vascular endothelial growth factor (VEGF), VEGF receptor Flk-1 and cyclooxygenase-2 (COX-2). Apoptosis was evaluated using the TUNEL assay. Hypothyroidism reduced the intensity and area of COX-2 expression in the corpus luteum (p < 0.05), while hyperthyroidism did not alter COX-2 expression in the dioestrus phase. Hypothyroidism significantly reduced the expression of CDC-47 in endothelial cells and pericytes in the corpus luteum, whereas hyperthyroidism did not induce a detectable change in CDC-47 expression (p > 0.05). Hypothyroidism reduced the level of apoptosis in luteal cells (p < 0.05) and increased VEGF expression in the corpus luteum. In contrast, hyperthyroidism increased the level of apoptosis in the corpus luteum (p < 0.05). In conclusion, thyroid dysfunction differentially affects the levels of proliferative activity, angiogenesis and apoptosis and COX-2 expression in the corpus luteum of female rats. © 2013 Blackwell Verlag GmbH.

Increased expression of 78 kD glucose-regulated protein promotes cardiomyocyte apoptosis in a rat model of liver cirrhosis

PubMed Central

Zhang, Lili; Zhang, Huiying; Lv, Minli; Jia, Jiantao; Fan, Yimin; Tian, Xiaoxia; Li, Xujiong; Li, Baohong; Ji, Jingquan; Wang, Limin; Zhao, Zhongfu; Han, Dewu; Ji, Cheng

2015-01-01

Aims: This study was to investigate the role and underlying mechanism of 78 kD glucose-regulated protein (GRP78) in cardiomyocyte apoptosis in a rat model of liver cirrhosis. Methods: A rat model of liver cirrhosis was established with multiple pathogenic factors. A total of 42 male SD rats were randomly divided into the liver cirrhosis group and control group. Cardiac structure analysis was performed to assess alterations in cardiac structure. Cardiomyocytes apoptosis was detected by TdT-mediated dUTP nick end labeling method. Expression of GRP78, CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) and B cell lymphoma-2 (Bcl-2) was detected by immunohistochemical staining. Results: The ratios of left ventricular wall thickness to heart weight and heart weight to body weight were significantly increased with the progression of liver cirrhosis (P < 0.05). Apoptosis index of cardiomyocytes was significantly increased with the progression of liver cirrhosis (P < 0.05). The expression levels of GRP78, CHOP and caspase-12 were significantly increased in the progression of liver cirrhosis (P < 0.05). The expression levels of NF-κB p65 and Bcl-2 were highest in the 4-wk liver cirrhosis, and they were decreased in the 6-wk and 8-wk in the progression of liver cirrhosis. GRP78 expression levels were positively correlated with apoptosis index, CHOP and caspase-12 expression levels (P < 0.05). CHOP expression levels were negatively correlated with NF-κB p65 and Bcl-2 expression levels (P < 0.05). Conclusion: Increased expression of GRP78 promotes cardiomyocyte apoptosis in rats with cirrhotic cardiomyopathy. PMID:26464674

Induction of apoptosis in MCF-7 cells by the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus Malaysian strain AF2240

PubMed Central

GHRICI, MOHAMED; EL ZOWALATY, MOHAMED; OMAR, ABDUL RAHMAN; IDERIS, AINI

2013-01-01

Newcastle disease virus (NDV) exerts its naturally occurring oncolysis possibly through the induction of apoptosis. We hypothesized that the binding of the virus to the cell via the hemagglutinin-neuraminidase (HN) glycoprotein may be sufficient to not only induce apoptosis but to induce a higher apoptosis level than the parental NDV AF2240 virus. NDV AF2240 induction of apoptosis in MCF-7 human breast cancer cells was analyzed and quantified. In addition, the complete HN gene of NDV strain AF2240 was amplified, sequenced and cloned into the pDisplay eukaryotic expression vector. HN gene expression was first detected at the cell surface membrane of the transfected MCF-7 cells. HN induction of apoptosis in transfected MCF-7 cells was analyzed and quantified. The expression of the HN gene alone was able to induce apoptosis in MCF-7 cells but it was a less potent apoptosis inducer compared to the parental NDV AF2240 strain. In conclusion, the NDV AF2240 strain is a more suitable antitumor candidate agent than its recombinant HN gene unless the latter is further improved by additional modifications. PMID:23807159

Induction of apoptosis in MCF-7 cells by the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus Malaysian strain AF2240.

PubMed

Ghrici, Mohamed; El Zowalaty, Mohamed; Omar, Abdul Rahman; Ideris, Aini

2013-09-01

Newcastle disease virus (NDV) exerts its naturally occurring oncolysis possibly through the induction of apoptosis. We hypothesized that the binding of the virus to the cell via the hemagglutinin-neuraminidase (HN) glycoprotein may be sufficient to not only induce apoptosis but to induce a higher apoptosis level than the parental NDV AF2240 virus. NDV AF2240 induction of apoptosis in MCF-7 human breast cancer cells was analyzed and quantified. In addition, the complete HN gene of NDV strain AF2240 was amplified, sequenced and cloned into the pDisplay eukaryotic expression vector. HN gene expression was first detected at the cell surface membrane of the transfected MCF-7 cells. HN induction of apoptosis in transfected MCF-7 cells was analyzed and quantified. The expression of the HN gene alone was able to induce apoptosis in MCF-7 cells but it was a less potent apoptosis inducer compared to the parental NDV AF2240 strain. In conclusion, the NDV AF2240 strain is a more suitable antitumor candidate agent than its recombinant HN gene unless the latter is further improved by additional modifications.

[Tryptase inhibits cell apoptosis through upregulating PAR-2 and Rho kinase in rheumatoid arthritis synovial fibroblasts].

PubMed

Zheng, Qianqian; Li, Shigang; Jia, Yunli; Liu, Wei; Yu, Lingling; Chen, Xianyong; Wang, Jinling

2016-12-01

Objective To investigate the regulatory effects of tryptase on protease-activated receptors 2 (PAR-2), Rho signal pathway and apoptosis of MH7A rheumatoid arthritis synovial fibroblasts. Methods In MH7A cells, the expression of PAR-2 was measured by flow cytometry; cell apoptosis was examined by annexin V- FITC/PI staining combined with flow cytometry; the expression of Rho kinase was detected by Pull-down assay and Western botting. Results Tryptase upregulated the expression of PAR-2 in MH7A cells, and suppressed Fas-mediated apoptosis of MH7A cells in a dose-dependent manner. Meanwhile, PAR-2 inhibitor, FSLLRY-NH2 significantly reduced anti-apoptotic effects of tryptase in MH7A cells, which was related with the increase of activated Rho kinase expression. Conclusion Tryptase plays a role in resistance to the apoptosis of MH7A cells through raising PAR-2 and activating Rho kinase.

Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis.

PubMed

Nutku, E; Zhuang, Q; Soussi-Gounni, A; Aris, F; Mazer, B D; Hamid, Q

2001-07-15

In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rbeta1 and -beta2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rbeta1 and -beta2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis.

Activation of Wnt3α/β-catenin signal pathway attenuates apoptosis of the cerebral microvascular endothelial cells induced by oxygen-glucose deprivation.

PubMed

Zhang, Jianshui; Zhang, Junfeng; Qi, Cunfang; Yang, Pengbo; Chen, Xinlin; Liu, Yong

2017-08-19

Brain microvascular endothelial cells (BMECs) play vital roles in cerebral ischemia, during which many signal pathways mediate BMECs apoptosis. In this study, we explored the potential role of Wnt3α/β-catenin signal in BMECs apoptosis induced by ischemia. Here, we found that oxygen-glucose deprivation (OGD) could induce apoptosis of BMECs with Wnt3a mRNA expression decrease. Meanwhile, activation Wnt3a/β-catenin signal with exogenous Wnt3α protein (100 ng/ml) or Lithium Chloride (LiCl, 4 mM) decreased significantly apoptosis of BMECs induced by OGD with increasing expression of Bcl-2 in the whole cell and β-catenin in the nucleus. But, inhibition Wnt3a/β-catenin signal with DKK1 (100 ng/ml) or 2.4-diamino quinazoline (DQ, 0.2 μM) increased apoptosis of BMECs with decreasing expression of Bcl-2. These results suggest that activation Wnt3α/β-catenin signal attenuate apoptosis of BMECs induced by ischemia. Copyright © 2017 Elsevier Inc. All rights reserved.

Apoptosis induction in duck tissues during duck hepatitis A virus type 1 infection.

PubMed

Sheng, X D; Zhang, W P; Zhang, Q R; Gu, C Q; Hu, X Y; Cheng, G F

2014-03-01

To investigate the role of apoptosis in duck viral hepatitis pathogenesis, 4- and 21-d-old ducks were inoculated with duck hepatitis A virus serotype 1 and killed at 2, 6, 12, 24, and 48 h postinfection. TdT-mediated dUTP nick-end labeling was used to detect apoptosis cells. Expression profiles of apoptosis-related genes including caspase-3, -8, -9, and Bcl-2 in spleen, bursa of Fabricius, liver, and the quantity of virus in blood were examined using real-time PCR. The TdT-mediated dUTP nick-end labeling analysis indicated there was a significant difference of apoptotic cells between treatments and controls. The same difference also appeared in virus amount variation in blood during infection. Gene expression analysis revealed that the apoptosis-related gene expression profile was different in the 2 groups, and also different between various organs. This study suggested that apoptosis may play an important role in duck hepatitis A virus serotype 1 infection, and apoptosis suppression might facilitate virus multiplication, resulting in the highest virus concentration in the host.

Human Embryonic and Induced Pluripotent Stem Cells Express TRAIL Receptors and Can Be Sensitized to TRAIL-Induced Apoptosis

PubMed Central

Vinarsky, Vladimir; Krivanek, Jan; Rankel, Liina; Nahacka, Zuzana; Barta, Tomas; Jaros, Josef; Andera, Ladislav

2013-01-01

Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis. PMID:23806100

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis

PubMed Central

Liu, Shuying; Zhang, Zhengyu; Wang, Jingjing; Zhou, Yuhuan; Liu, Kefeng; Huang, Jintao; Chen, Dadi; Wang, Junmei; Li, Chaohong

2015-01-01

Aims This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms. Methods Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression. Results Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis. Conclusions Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels. PMID:26488175

Nature and mechanisms of hepatocyte apoptosis induced by D-galactosamine/lipopolysaccharide challenge in mice.

PubMed

Wu, Yi-Hang; Hu, Shao-Qing; Liu, Jun; Cao, Hong-Cui; Xu, Wei; Li, Yong-Jun; Li, Lan-Juan

2014-06-01

Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6-10 h after the intraperitoneal injection of D-GalN (700 mg/kg) and LPS (10 µg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering D-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end‑labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following D-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the D-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by D-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis.

Experimental variation of the level and the ratio of angiogenic and osteogenic signaling affects the spatiotemporal expression of bone-specific markers and organization of bone formation in ectopic sites.

PubMed

Moser, Norman; Goldstein, Jan; Kauffmann, Phillip; Epple, Matthias; Schliephake, Henning

2018-04-01

The aim of the present study was to test the hypothesis that the ratio of angiogenic and osteogenic signaling affects ectopic bone formation when delivered in different amounts. Porous composite PDLLA/CaCO 3 scaffolds were loaded with rhBMP2 and rhVEGF in different dosage combinations and implanted into the gluteal muscles of 120 adult male Wistar rats. Bone formation and expression of alkaline phosphatase and Runx2 were quantified by histomorphometry. Spatial distribution across the scaffolds was assessed by using a grid that discriminated between the periphery and center of the scaffolds. The evaluation showed that the combined delivery of bone morphogenetic protein BMP2 and VEGF in different dosage combinations did not enhance the overall quantity of ectopic bone formation compared to the delivery of BMP2 alone. The addition of VEGF generally upregulated Runx2 after 4 weeks, which may have retarded terminal osteogenic differentiation. However, slow combined delivery of 1.5-2.0 μg BMP2 combined with 50 ng VEGF165 over a period of 5 weeks supported a more even distribution of bone formation across the implanted scaffolds whereas higher amounts of VEGF did not elicit this effect. The findings suggest that structural organization rather than the quantity of ectopic bone formation is affected by the dosage and the ratio of BMP2 and VEGF levels at the observed intervals. The development of carriers for dual growth factor delivery has to take into account the necessity to carefully balance the ratio of growth release.