DOCSLIB.ORG

High Efficacy of Fibroblast and Hepatocyte Growth Factors on the in Vitro Blastocyst Production of Post- Thaw Mouse Two-Cell Embryo: the Cryotop Method

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
High Efficacy of Fibroblast and Hepatocyte Growth Factors on the in Vitro Blastocyst Production of Post- Thaw Mouse Two-Cell Embryo: the Cryotop Method frican Journal of Biotechnology Vol. 10(69), pp. 15667-15672, 7 November, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.446 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper High efficacy of fibroblast and hepatocyte growth factors on the in vitro blastocyst production of post- thaw mouse two-cell embryo: The cryotop method Fatemeh Ghasemian1, Mahnaz Azarnia2, Abtin Heidarzadeh3, Shervin Ghadarjani4 and Mohammad Hadi Bahadori5* 1Department of Biology, Faculty of Science, Kharazmi (Tarbiat moallem) University, Tehran, Iran. 2Department of Biology, Faculty of Science, Kharazmi (Tarbiat moallem) University, Tehran, Iran. 3Department of Community Medicine, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran. 4Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran. 5Cellular and Molecular Research Center, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran. Accepted 5 September, 2011 There is great need to improve our understanding of what increases an embryo’s development potential, after vitrification-thawing processes. For this subject, 358 two-cell stage embryos were collected from oviduct of pregnant two-day old mice and vitrified. After thawing, embryos were cultured in Tyrode's (T6) medium supplemented with different doses of fibroblast growth factor (FGF; 0, 10, 20, 50 and 100 ng/ml) and hepatocyte growth factor (HGF; 0, 10, 20, 50 and 100 ng/ml) until the blastocyst stage. To determine quality of blastocysts, blastocysts were stained with hoechst and propidium iodide. After culture for 24 h, 92.65% of treated embryos with 20 ng/ml of FGF had higher (P<0.05) survival rate in comparison to the control group (84%). Blastocyst embryo formation rate were 79.43% (P< 0.01), and 67.46% (P< 0.05) in the treated groups with 20 ng/ml of FGF and 20 ng/ml of HGF, respectively, which were significantly different from the control (56%). In the treatment group with 20 ng/ml of FGF, blastocysts with >64 cells had a significantly higher inner cell mass (ICM) in comparison to the control group (P< 0.01). In conclusion, in this experiment, addition of growth factors in the culture had favorable effects on post-thawed cleavage of vitrified 2-cell embryos and blastocyst quality. Key words: Blastocyst quality, growth factors, preimplantation development, vitrification. INTRODUCTION Following scientific efforts, more logical and economical of assisted reproduction techniques are discovered. One procedures in order to maintain and promote the success of these procedures is cryopreservation of oocytes, zygotes and embryos (Al-Hasani et al., 2007). The first report about successful frozen-thawed human embryos was in the early 1980s (Al-Hasani et al., 2007). Vitrifi- cation with a rapid cooling rate and high concentrations of *Corresponding author. E-mail: [email protected]. Tel: +98- 131-6690884 or 6690099.Fax: +98- 131-6690007. CPAs could be used. Complete deletion of ice crystal formation (Bahadori et al., 2009; Kasai and Mukaida, 2004; Vajta and Nagy, 2006), spending a shorter time, Abbreviations: FGF, Fibroblast growth factor; HGF, hepatocyte growth factor; hCG, human chorionic gonadotrophin; ART, independency to a programmable temperature container assisted reproductive techniques; TE, trophoectoderm; ICM, (Karlsson, 2002), its feasibility (Zhang et al., 2009) and inner cell mass; GUMS, Guilan university of medical science; handleability by only one embryologist (Kuwayama et al., PMSG, gonadotrophin; EG, ethylene glycol; DMSO, dimethyl 2005b) are the other advantages of this method. sulphoxide; TCN, total cell number. Recently, changes in the culture systems and cryo- 15668 Afr. J. Biotechnol. preservation methods appear to improve in vitro MATERIALS AND METHODS production of cryopreserved embryos (Khurana and Animals and embryo recovery Niemann, 2000; Mtango et al., 2003). There is great need to improve our understanding of what increases an All of the reagents were purchased from Sigma (Germany) except embryo’s development potential, after vitrification-thawing hCG that was purchased from Organon (Holand), HGF and bFGF-4 process. A few studies were performed with FGF and from R and D systems (USA), Cryotop from Kitazato Company HGF. FGFs are potent physiological regulators of growth (Japan) and NMRI mice from the Razi Institute of Karaj (Iran). 30 female mice aged six to eight weeks old were kept under the and proliferation of the cells (Detillieux et al., 2003) and it controlled light and temperature conditions with free access to is known to be a powerful mitogen for granulosa cells of water and food. All animals were treated in accordance with the various species in culture (Quennell et al., 2004). guidelines of the Guilan University of Medical Science (GUMS) However, Bieser et al. (1998) reported that the role of Ethics Community Standards on the Care and Use of Laboratory Animals. They were super-ovulated by intra-peritoneal injection of 5 modulating growth factor like FGF was essential for ’ extra-cellular proteolysis occurring during in vitro IU of pregnant mare s serum gonadotrophin (PMSG), and followed by injection of 5 IU of hCG for 48 h. On the same day of hCG maturation of cumulus oocyte complexes. FGF is key- injection, designated as day 0, the female mice were paired with players in the processes of proliferation and differen- male mice of the same strain to mate. The mated females were tiation of oocytes (Pandey et al., 2009). But, a few then killed 48 h after hCG administration and two-cell stage studied has been shown the effect of FGF as proliferation embryos (Figure 1a) were harvested from the oviducts in pre- agent in embryo. Nandi et al. (2003) observed that FGF warmed T6 medium. After washing several times, the obtained 2- cell embryos with the normal morphology were prepared for significantly increased cleavage and blastocysts rates of vitrification. buffalo embryos as compared to the control. Several lines of studies have investigated HGF in mitogenic, motogenic and morphogenic functions on Vitrification and thawing procedures various epithelial cells derived from rodents and human The vitrification/thawing protocol was performed according to the (Osuga et al., 1999). It has been reported that HGF is method described by kuwayama (2007). The two-cell stage embryo present in follicular fluid and its expression is stimulated were incubated in equilibration solution comprising 7.5% ethylene by human chorionic gonadotrophin (hCG) (Osuga et al., glycol (EG) and 7.5% dimethyl sulphoxide (DMSO) in Ham’s F-10 1999). It has been shown that FGF stimulate HGF media for 5 to15 min (depending on the time needed for re- expression in non-reproductive cells and tissues (Osuga expansion of the cell) at room temperature. After an initial shrinkage and recovery, they were then aspirated and placed into the et al., 1999). HGF-induced signaling motifs coupled to the vitrification solution (15% EG, 15% DMSO, 0.5 M sucrose) in Ham’s regulation of cell cycle progression, morphogenesis and F-10 medium for 50 to 60 s at room temperature. As soon as cell survival (Zachow and Uzumcu, 2007). So, that might cellular shrinkage was absorbed (Figure 1b), embryos were impress proliferation, cell cycle progression and survival aspirated and placed on the tip of the Cryotop (Figure 1c). Cooling of embryos. of the embryos was done by direct contact with liquid nitrogen. The The need for efficient cryopreservation of oocytes and Cryotops were stored in liquid nitrogen for at least two weeks. Warming of embryos was performed by placing the Cryotop in embryos is enormous both for theoretical and practical thawing solution (1 M sucrose) for 50 to 60s at room temperature reasons. However, in spite of the vast efforts invested, and then into dilution solution (0.5 M sucrose) for 3 min, followed by advances are rather slow. The main problems are lack of another dilution solution of 0.25 M sucrose for another 3 min, both consistency as well as differences in survival and at room temperature. The warmed embryos were placed four to five developmental rates after warming between species, times into washing solution (Ham’s F-10) before incubation. The embryo quality and survival rate were assessed morphologically. developmental stages and quality (Kuwayama, 2007). The suboptimal culture condition in which fertilization and early embryonic growth occur affected the success rates Fibroblast growth factor and hepatocyte growth factor of assisted reproductive techniques (ART). There are the problems such as embryo fragmentation, cell irregularity, To demonstrate any positive effects of growth factors supple- mentation in culture medium on survival and development after reduction in total cell numbers, retarded cleavage rate thawing, thawed two-cell stage embryos were incubated for 72 h in and blastulation in in vitro blastocysts growth, attributed medium at 37°C in a humidified 5% CO2 air as the following to suboptimal in vitro conditions, which in turn contribute treatment groups: T6 + 5mg/ml of bovine serum albumin (BSA) significantly to reduce embryo quality and implantation (control), T6 + 5mg/ml of BSA + different doses of FGF (1 to 4 rate following embryo transfer (Cheung et al., 2003). The treated groups: 10, 20, 50 and 100 ng/ml, respectively) and T6 + 5 aim of this study was to determine whether the exposure mg/ml of BSA + different doses of HGF (1 to 4 treated groups: 10, 20, 50 and 100 ng/ml, respectively). The embryos survival was of embryos to growth factors prior to implantation, would defined as the percentage of vitrified embryos that re-expanded affect thawed embryo development rates. As a second after 24 h. The thawed embryos were cultured until blastocyst stage. objective, total cell number, TE (trophoectoderm) and ICM were assessed to determine whether the growth Blastocyst cell number determination factors can promote the reduced cell number in blasto- cysts derived from vitrified mouse embryos. To determine blastocyst cell numbers (vitrified and non-vitrified) Ghasemian et al.
Load more

Recommended publications
  • Review Article Receptor Tyrosine Kinases in Kidney Development
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Hindawi Publishing Corporation Journal of Signal Transduction Volume 2011, Article ID 869281, 10 pages doi:10.1155/2011/869281 Review Article Receptor Tyrosine Kinases in Kidney Development Renfang Song, Samir S. El-Dahr, and Ihor V. Yosypiv Section of Pediatric Nephrology, Department of Pediatrics, Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA Correspondence should be addressed to Ihor V. Yosypiv, [email protected] Received 25 November 2010; Revised 8 January 2011; Accepted 15 January 2011 Academic Editor: Karl Matter Copyright © 2011 Renfang Song et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The kidney plays a fundamental role in the regulation of arterial blood pressure and fluid/electrolyte homeostasis. As congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most common human birth defects, improved understanding of the cellular and molecular mechanisms that lead to CAKUT is critical. Accumulating evidence indicates that aberrant signaling via receptor tyrosine kinases (RTKs) is causally linked to CAKUT. Upon activation by their ligands, RTKs dimerize, undergo autophosphorylation on specific tyrosine residues, and interact with adaptor proteins to activate intracellular signal transduction pathways that regulate diverse cell behaviours such as cell proliferation, survival, and movement. Here, we review the current understanding of role of RTKs and their downstream signaling pathways in the pathogenesis of CAKUT.
    [Show full text]
  • Gene Expression Analysis Identifies Potential Biomarkers of Neurofibromatosis Type 1 Including Adrenomedullin
    Published OnlineFirst August 25, 2010; DOI: 10.1158/1078-0432.CCR-10-0613 Clinical Imaging, Diagnosis, Prognosis Cancer Research Gene Expression Analysis Identifies Potential Biomarkers of Neurofibromatosis Type 1 Including Adrenomedullin Trent R. Hummel1, Walter J. Jessen1, Shyra J. Miller1, Lan Kluwe4, Victor F. Mautner4, Margaret R. Wallace5, Conxi Lázaro6, Grier P. Page7, Paul F. Worley8, Bruce J. Aronow2, Elizabeth K. Schorry3, and Nancy Ratner1 Abstract Purpose: Plexiform neurofibromas (pNF) are Schwann cell tumors found in a third of individuals with neurofibromatosis type 1 (NF1). pNF can undergo transformation to malignant peripheral nerve sheath tumors (MPNST). There are no identified serum biomarkers of pNF tumor burden or transformation to MPNST. Serum biomarkers would be useful to verify NF1 diagnosis, monitor tumor burden, and/or detect transformation. Experimental Design: We used microarray gene expression analysis to define 92 genes that encode putative secreted proteins in neurofibroma Schwann cells, neurofibromas, and MPNST. We validated dif- ferential expression by quantitative reverse transcription-PCR, Western blotting, and ELISA assays in cell conditioned medium and control and NF1 patient sera. Results: Of 13 candidate genes evaluated, only adrenomedullin (ADM) was confirmed as differentially expressed and elevated in serum of NF1 patients. ADM protein concentrati on was further elevated in serum of a small sampling of NF1 patients with MPNST. MPNST cell conditioned medium, containing ADM and hepatocyte growth factor, stimulated MPNST migration and endothelial cell proliferation. Conclusions: Thus, microarray analysis identifies potential serum biomarkers for disease, and ADM is a serum biomarker of NF1. ADM serum levels do not seem to correlate with the presence of pNFs but may be a biomarker of transformation to MPNST.
    [Show full text]
  • Different Fgfs Have Distinct Roles in Regulating Neurogenesis After Spinal Cord Injury in Zebrafish Yona Goldshmit1,2, Jean Kitty K
    Goldshmit et al. Neural Development (2018) 13:24 https://doi.org/10.1186/s13064-018-0122-9 RESEARCHARTICLE Open Access Different Fgfs have distinct roles in regulating neurogenesis after spinal cord injury in zebrafish Yona Goldshmit1,2, Jean Kitty K. Y. Tang1, Ashley L. Siegel1, Phong D. Nguyen1, Jan Kaslin1, Peter D. Currie1 and Patricia R. Jusuf1,3* Abstract Background: Despite conserved developmental processes and organization of the vertebrate central nervous system, only some vertebrates including zebrafish can efficiently regenerate neural damage including after spinal cord injury. The mammalian spinal cord shows very limited regeneration and neurogenesis, resulting in permanent life-long functional impairment. Therefore, there is an urgent need to identify the cellular and molecular mechanisms that can drive efficient vertebrate neurogenesis following injury. A key pathway implicated in zebrafish neurogenesis is fibroblast growth factor signaling. Methods: In the present study we investigated the roles of distinctfibroblastgrowthfactormembersandtheir receptors in facilitating different aspects of neural development and regeneration at different timepoints following spinal cord injury. After spinal cord injury in adults and during larval development, loss and/or gain of Fgf signaling was combined with immunohistochemistry, in situ hybridization and transgenes marking motor neuron populations in in vivo zebrafish and in vitro mammalian PC12 cell culture models. Results: Fgf3 drives neurogenesis of Islet1 expressing motor neuron subtypes and mediate axonogenesis in cMet expressing motor neuron subtypes. We also demonstrate that the role of Fgf members are not necessarily simple recapitulating development. During development Fgf2, Fgf3 and Fgf8 mediate neurogenesis of Islet1 expressing neurons and neuronal sprouting of both, Islet1 and cMet expressing motor neurons.
    [Show full text]
  • Artificial Liver Support Potential to Retard Regeneration?
    REVIEW ARTICLE Artificial Liver Support Potential to Retard Regeneration? Emma J. Mullin, MBChB; Matthew S. Metcalfe, FRCS; Guy J. Maddern, MD Hypothesis: The concept of an “artificial liver” has been growth-promoting factors from these cultured hepato- in development for over 40 years. Such devices aim to cytes? temporarily assume metabolic and excretory functions of the liver, with removal of potentially hepatotoxic sub- Data Sources, Extraction, and Study Selection: stances, thereby clinically stabilizing patients and pre- Data were obtained using PubMed search for reports in- venting deterioration while awaiting transplantation. If volving liver support, extracorporeal circuits, dialysis, sufficient numbers of viable hepatocytes remain, regen- growth factors, and cytokines. Those reports specifi- eration and subsequent recovery of innate liver func- cally looking at the effect of artificial liver support on cy- tion may occur. However, these devices have not yet be- tokines and growth factors are discussed. come part of routine clinical use. Much less is known regarding the effect such devices have, if any, on circu- Conclusions: There is a paucity of information on the lating cytokines and growth factors and the subsequent key events and substances involved in hepatic regenera- effects on the regenerating liver. If these devices remove tion. In addition, there is a potential impact of liver sup- or reduce factors known to promote regeneration, is the port devices on the regeneration of substances associ- rate of regeneration retarded? Conversely, does the in- ated with hepatic regeneration. Further study is needed. corporation of hepatocytes into bioartificial support sys- tems confer an advantage through the production of Arch Surg.
    [Show full text]
  • Hepatocyte Growth Factor Signaling Pathway As a Potential Target in Ductal Adenocarcinoma of the Pancreas
    JOP. J Pancreas (Online) 2017 Nov 30; 18(6):448-457. REVIEW ARTICLE Hepatocyte Growth Factor Signaling Pathway as a Potential Target in Ductal Adenocarcinoma of the Pancreas Samra Gafarli, Ming Tian, Felix Rückert Department of Surgery, Medical Faculty Mannheim, University of Heidelberg, Germany ABSTRACT Hepatocyte growth factor is an important cellular signal pathway. The pathway regulates mitogenesis, morphogenesis, cell migration, invasiveness and survival. Hepatocyte growth factor acts through activation of tyrosine kinase receptor c-Met (mesenchymal epithelial transition factor) as the only known ligand. Despite the fact that hepatocyte growth factor is secreted only by mesenchymal origin cells, the targets of this multifunctional pathway are cells of mesenchymal as well as epithelial origin. Besides its physiological role recent evidences suggest that HGF/c-Met also plays a role in tumor pathophysiology. As a “scatter factor” hepatocyte growth factor stimulates cancer cell migration, invasion and subsequently promote metastases. Hepatocyte growth factor further is involved in desmoplastic reaction and consequently indorse chemo- and radiotherapy resistance. Explicitly, this pathway seems to mediate cancer cell aggressiveness and to correlate with poor prognosis and survival rate. Pancreatic Ductal Adenocarcinoma is a carcinoma with high aggressiveness and metastases rate. Latest insights show that the HGF/c-Met signal pathway might play an important role in pancreatic ductal adenocarcinoma pathophysiology. In the present review, we highlight the role of HGF/c-Met pathway in pancreatic ductal adenocarcinoma with focus on its effect on cellular pathophysiology and discuss its role as a potential therapeutic target in pancreatic ductal adenocarcinoma. INTRODUCTION activation causes auto-phosphorylation of c-Met and subsequent activation of downstream signaling pathways Hepatocyte growth factor (HGF) is a multifunctional such as mitogen-activated protein kinases (MAPKs), gene.
    [Show full text]
  • Hepatocyte Growth Factor: a Regulator of Inflammation and Autoimmunity
    Autoimmunity Reviews 14 (2015) 293–303 Contents lists available at ScienceDirect Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev Review Hepatocyte growth factor: A regulator of inflammation and autoimmunity Nicolas Molnarfi a,b,1, Mahdia Benkhoucha a,b,1, Hiroshi Funakoshi d, Toshikazu Nakamura e, Patrice H. Lalive a,b,c,⁎ a Department of Pathology and Immunology, Faculty of Medicine, University of Geneva, Geneva, Switzerland b Department of Clinical Neurosciences, Division of Neurology, Unit of Neuroimmunology and Multiple Sclerosis, University Hospital of Geneva, Geneva, Switzerland c Department of Genetics and Laboratory Medicine, Laboratory Medicine Service, University Hospital of Geneva, Geneva, Switzerland d Center for Advanced Research and Education, Asahikawa Medical University, Asahikawa, Japan e Neurogen Inc., Nakahozumi, Ibaraki, Osaka, Japan article info abstract Article history: Hepatocyte growth factor (HGF) is a pleiotropic cytokine that has been extensively studied over several decades, Received 20 November 2014 but was only recently recognized as a key player in mediating protection of many types of inflammatory and au- Accepted 25 November 2014 toimmune diseases. HGF was reported to prevent and attenuate disease progression by influencing multiple Available online 1 December 2014 pathophysiological processes involved in inflammatory and immune response, including cell migration, matura- tion, cytokine production, antigen presentation, and T cell effector function. In this review, we discuss the actions Keywords: fl HGF and mechanisms of HGF in in ammation and immunity and the therapeutic potential of this factor for the treat- fl c-Met ment of in ammatory and autoimmune diseases. Inflammation © 2014 Elsevier B.V. All rights reserved. Autoimmunity Autoimmune regulator Therapy Contents 1.
    [Show full text]
  • The Role of Hepatocyte Growth Factor in Mesenchymal Stem Cell-Induced
    Song et al. Stem Cell Research & Therapy (2020) 11:178 https://doi.org/10.1186/s13287-020-01691-x RESEARCH Open Access The role of hepatocyte growth factor in mesenchymal stem cell-induced recovery in spinal cord injured rats Peiwen Song1†, Tianyu Han1†, Xia Xiang1, Ying Wang2, Huang Fang3, Yang Niu1 and Cailiang Shen1* Abstract Background: Mesenchymal stem cells (MSCs) have become a promising treatment for spinal cord injury (SCI) due to the fact that they provide a favorable environment. Treatment using MSCs results in a better neurological functional improvement through the promotion of nerve cell regeneration and the modulation of inflammation. Many studies have highlighted that the beneficial effects of MSCs are more likely associated with their secreted factors. However, the identity of the factor that plays a key role in the MSC-induced neurological functional recovery following SCI as well as its molecular mechanism still remains unclear. Methods: A conditioned medium (collected from the MSCs) and hepatocyte growth factor (HGF) were used to test the effects on the differentiation of neural stem cells (NSCS) in the presence of BMP4 with or without a c-Met antibody. In SCI rats, Western blot, ELISA, immunohistochemistry, and hematoxylin-eosin staining were used to investigate the biological effects of MSC-conditioned medium and HGF on nerve cell regeneration and inflammation with or without the pre-treatment using a c-Met antibody. In addition, the possible molecular mechanism (cross-talk between HGF/c-Met and the BMP/Smad 1/5/8 signaling pathway) was also detected by Western blot both in vivo and in vitro.
    [Show full text]
  • In Vitro Differentiation of Human Umbilical Cord Blood Mesenchymal
    Alexandria Journal of Medicine (2017) 53, 167–173 HOSTED BY Alexandria University Faculty of Medicine Alexandria Journal of Medicine http://www.elsevier.com/locate/ajme In vitro differentiation of human umbilical cord blood mesenchymal stem cells into functioning hepatocytes May H. Hasan a, Kamal G. Botros a, Mona A. El-Shahat a,*, Hussein A. Abdallah b, Mohamed A. Sobh c a Anatomy and Embryology Department, Faculty of Medicine, Mansoura University, Egypt b Medical Biochemistry Department, Faculty of Medicine, Mansoura University, Egypt c Experimental Biology, Urology & Nephrology Center, Mansoura University, Egypt Received 29 January 2016; revised 6 May 2016; accepted 14 May 2016 Available online 5 August 2016 KEYWORDS Abstract Mesenchymal stem cells (MSCs) were isolated by gradient density centrifugation from Umbilical cord blood; umbilical cord blood. Spindle-shaped adherent cells were permitted to grow to 70% confluence Mesenchymal stem cells; in primary culture media which was reached by day 12. Induction of differentiation started by cul- Culture; turing cells with differentiation medium containing FGF-4 and HGF. Under hepatogenic condi- Hepatocytes; tions few cuboidal cells appeared in culture on day 7. From day 21 to day 28, most of cells HGF; became small and round. The control negative cells cultured in serum free media showed FGF-4 fibroblast-like morphology. Urea production and protein secretion by the differentiated hepatocyte-like cells were detected on day 21 and increased on day 28. Protein was significantly increased in comparison with control by day 28. The cells became positive for AFP at day 7 and positive cells could still be detected at days 21 and 28.
    [Show full text]
  • Migration Dichotomy of Glioblastoma by Interacting with Focal Adhesion Kinase
    Oncogene (2012) 31, 5132 --5143 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase SD Wang1, P Rath1,2, B Lal1,2, J-P Richard1,2,YLi1,2, CR Goodwin3, J Laterra1,2,4,5 and S Xia1,2 Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumors in adults. Uncontrolled proliferation and abnormal cell migration are two prominent spatially and temporally disassociated characteristics of GBMs. In this study, we investigated the role of the receptor tyrosine kinase EphB2 in controlling the proliferation/migration dichotomy of GBM. We studied EphB2 gain of function and loss of function in glioblastoma-derived stem-like neurospheres, whose in vivo growth pattern closely replicates human GBM. EphB2 expression stimulated GBM neurosphere cell migration and invasion, and inhibited neurosphere cell proliferation in vitro. In parallel, EphB2 silencing increased tumor cell proliferation and decreased tumor cell migration. EphB2 was found to increase tumor cell invasion in vivo using an internally controlled dual-fluorescent xenograft model. Xenografts derived from EphB2-overexpressing GBM neurospheres also showed decreased cellular proliferation. The non-receptor tyrosine kinase focal adhesion kinase (FAK) was found to be co-associated with and highly activated by EphB2 expression, and FAK activation facilitated focal adhesion formation, cytoskeleton structure change and cell migration in EphB2-expressing GBM neurosphere cells. Taken together, our findings indicate that EphB2 has pro-invasive and anti-proliferative actions in GBM stem-like neurospheres mediated, in part, by interactions between EphB2 receptors and FAK.
    [Show full text]
  • Hepatocyte Growth Factor Promotes Hepatocarcinogenesis Through C-Met Autocrine Activation and Enhanced Angiogenesis in Transgenic Mice Treated with Diethylnitrosamine
    Oncogene (2002) 21, 1791 ± 1799 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc ORIGINAL PAPERS Hepatocyte growth factor promotes hepatocarcinogenesis through c-Met autocrine activation and enhanced angiogenesis in transgenic mice treated with diethylnitrosamine Norio Horiguchi1, Hisashi Takayama1, Mitsuo Toyoda1, Toshiyuki Otsuka1, Toshio Fukusato2, Glenn Merlino3, Hitoshi Takagi*,1 and Masatomo Mori1 1The First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; 2Division of Diagnostic Pathology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; 3Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland, MD 20892-4255, USA Hepatocyte growth factor (HGF) is a mitogen for Introduction hepatocytes, but it is not clear whether HGF stimulates or inhibits hepatocarcinogenesis. We previously reported Hepatocyte growth factor (HGF) is a polypeptide that HGF transgenic mice under the metallothionein originally characterized as a highly potent hepatocyte gene promoter developed benign and malignant liver mitogen (Gohda et al., 1988; Nakamura et al., 1989). tumors spontaneously after 17 months of age. To More recent studies have revealed that HGF is a elucidate the role of HGF in hepatocarcinogenesis, multifunctional cytokine and can elicit mitogenic, diethylnitrosamine (DEN) was administered to HGF motogenic, and morphogenic responses in a variety of transgenic mice. HGF overexpression accelerated DEN- cultured
    [Show full text]
  • Table S1. List of All Proteins Included in the Proseek® Multiplex Oncology I V2 96X96 Cancer Panel
    Table S1. List of all proteins included in the Proseek® Multiplex Oncology I v2 96x96 Cancer Panel. Adrenomedullin (AM) Ezrin (EZR) Latency-associated peptide transforming growth factor beta-1 (LAP TGF-beta-1) Amphiregulin (AR) Fas antigen ligand (FasL) Angiopoietin-1 receptor (TIE2) FAS-associated death domain protein (FADD) Lipopolysaccharide-induced tumor necrosis factor- alpha factor (LITAF) B-cell activating factor (BAFF) Fms-related tyrosine kinase 3 ligand (Flt3L) Cadherin-3 (CDH3) Folate receptor alpha (FR-alpha) Macrophage colony-stimulating factor 1 (CSF-1) Carbonic anhydrase IX (CAIX) Follistatin (FS) Matrix metalloproteinase-1 (MMP-1) Carcinoembryonic antigen (CEA) Furin (FUR) Melanoma-derived growth regulatory protein (MIA) Caspase-3 (CASP-3) Growth hormone (GH) MHC class I polypeptide-related sequence A (MIC-A) C-C motif chemokine 19 (CCL19) Growth/differentiation factor 15 (GDF-15) Midkine (MK) CD40 ligand (CD40-L) Heparin-binding EGF-like growth factor (HB-EGF) Monocyte chemotactic protein 1 (MCP-1) C-X-C motif chemokine 5 (CXCL5) Hepatocyte growth factor (HGF) Myeloid differentiation primary response protein MyD88 (MYD88) C-X-C motif chemokine 9 (CXCL9) ICOS ligand (ICOSLG) C-X-C motif chemokine 10 (CXCL10) Immunoglobulin-like transcript 3 (ILT-3) NF-kappa-B essential modulator (NEMO) C-X-C motif chemokine 11 (CXCL11) Integrin alpha-1 (ITGA1) NT-3 growth factor receptor (NTRK3) C-X-C motif chemokine 13 (CXCL13) Interferon gamma (IFN-gamma) Ovarian cancer-related tumor marker CA 125 (CA-125) Cyclin-dependent kinase inhibitor
    [Show full text]
  • Hepatocyte Growth Factor Incorporated Into Herpes Simplex Virus Vector Accelerates Facial Nerve Regeneration After Crush Injury
    Gene Therapy (2011) 18, 1063–1069 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt ORIGINAL ARTICLE Hepatocyte growth factor incorporated into herpes simplex virus vector accelerates facial nerve regeneration after crush injury S Esaki1,2, J Kitoh3, S Katsumi1, F Goshima2, H Kimura2, M Safwat1, K Yamano1, N Watanabe1, N Nonoguchi4, T Nakamura5, RS Coffin6, S-I Miyatake4, Y Nishiyama2 and S Murakami1 Hepatocyte growth factor (HGF) promotes regeneration of the central nervous system, but its effects on the peripheral nervous system remain unclear. This study was conducted to elucidate the effect of HGF on regeneration of the murine facial nerve after crush injury. To do so, a replication-defective herpes simplex virus vector that incorporated HGF was prepared (HSV-HGF). The main trunk of the facial nerve was compressed by mosquito hemostats, and HSV-HGF, control vector or medium was then applied to the compressed nerve. We found that mice in the HGF group required significantly fewer days for complete recovery from nerve compression. Furthermore, the amplitude of the evoked buccinator muscle compound action potential increased following HSV-HGF application. HGF expression in and around the compressed nerve was demonstrated by enzyme-linked immunoassay and immunohistochemistry. In addition, HSV-HGF introduction around the damaged nerve significantly accelerated recovery of function of the facial nerve. These data suggest a possible role of HGF in promoting facial nerve regeneration after nerve damage. Furthermore, this viral delivery method may be applied clinically for many types of severe facial palsy during facial nerve decompression surgery. Gene Therapy (2011) 18, 1063–1069; doi:10.1038/gt.2011.57; published online 12 May 2011 Keywords: facial nerve injury; hepatocyte growth factor; HSV INTRODUCTION promote peripheral nerve regeneration; however, little is known about Numerous studies have used the facial nerve axotomy model to its effects on the peripheral nerve systems.
    [Show full text]