|
1
|
Divashuk MG, Nikitina EA, Sokolova VM, Yurkina AI, Kocheshkova AA, Razumova OV, Karlov GI, Kroupin PY. qPCR as a Selective Tool for Cytogenetics. PLANTS (BASEL, SWITZERLAND) 2022; 12:80. [PMID: 36616209 PMCID: PMC9824742 DOI: 10.3390/plants12010080] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Revised: 12/18/2022] [Accepted: 12/20/2022] [Indexed: 06/17/2023]
Abstract
qPCR is widely used in quantitative studies of plant genomes and transcriptomes. In this article, this method is considered as an auxiliary step in the preparation and selection of markers for FISH analysis. Several cases from the authors' research on populations of the same species were reviewed, and a comparison of the closely related species, as well as the adaptation of the markers, based on satellite tandem repeats (TRs) using quantitative qPCR data was conducted. In the selected cases, TRs with contrast abundance were identified in the cases of the Dasypyrum, Thinopyrum and Aegilops species, and the transfer of TRs between the wheat and related species was demonstrated. TRs with intraspecific copy number variation were revealed in Thinopyrum ponticum and wheat-wheatgrass partial amphidiploids, and the TR showing predominant hybridization to the sea buckthorn Y chromosome was identified. Additionally, problems such as the absence of a reference gene for qPCR, and low-efficiency and self-complementary primers, were illustrated. In the cases considered here, the qPCR results clearly show high correlation with the subsequent results of the FISH analysis, which confirms the value of this method for cytogenetic studies.
Collapse
|
|
2
|
Helweg LP, Storm J, Witte KE, Schulten W, Wrachtrup L, Janotte T, Kitke A, Greiner JFW, Knabbe C, Kaltschmidt B, Simon M, Kaltschmidt C. Targeting Key Signaling Pathways in Glioblastoma Stem Cells for the Development of Efficient Chemo- and Immunotherapy. Int J Mol Sci 2022; 23:12919. [PMID: 36361720 PMCID: PMC9659205 DOI: 10.3390/ijms232112919] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2022] [Revised: 09/23/2022] [Accepted: 10/21/2022] [Indexed: 01/12/2024] Open
Abstract
Glioblastoma multiforme (GBM) is the most aggressive and most common malignant brain tumor with poor patient survival despite therapeutic intervention. On the cellular level, GBM comprises a rare population of glioblastoma stem cells (GSCs), driving therapeutic resistance, invasion, and recurrence. GSCs have thus come into the focus of therapeutic strategies, although their targeting remains challenging. In the present study, we took advantage of three GSCs-populations recently established in our lab to investigate key signaling pathways and subsequent therapeutic strategies targeting GSCs. We observed that NF-κB, a crucial transcription factor in GBM progression, was expressed in all CD44+/CD133+/Nestin+-GSC-populations. Exposure to TNFα led to activation of NF-κB-RELA and/or NF-κB-c-REL, depending on the GBM type. GSCs further expressed the proto-oncogene MYC family, with MYChigh GSCs being predominantly located in the tumor spheres ("GROW"-state) while NF-κB-RELAhigh GSCs were migrating out of the sphere ("GO"-state). We efficiently targeted GSCs by the pharmacologic inhibition of NF-κB using PTDC/Bortezomib or inhibition of MYC by KJ-Pyr-9, which significantly reduced GSC-viability, even in comparison to the standard chemotherapeutic drug temozolomide. As an additional cell-therapeutic strategy, we showed that NK cells could kill GSCs. Our findings offer new perspectives for developing efficient patient-specific chemo- and immunotherapy against GBM.
Collapse
Affiliation(s)
- Laureen P. Helweg
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
| | - Jonathan Storm
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
| | - Kaya E. Witte
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
| | - Wiebke Schulten
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
| | - Lennart Wrachtrup
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
| | - Till Janotte
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
| | - Angelika Kitke
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
| | - Johannes F. W. Greiner
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
| | - Cornelius Knabbe
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
- Heart and Diabetes Centre NRW, Institute for Laboratory and Transfusion Medicine, Ruhr-University Bochum, 32545 Bad Oeynhausen, Germany
| | - Barbara Kaltschmidt
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
- Molecular Neurobiology, Faculty of Biology, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany
| | - Matthias Simon
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
- Department of Neurosurgery and Epilepsy Surgery, Protestant Hospital of Bethel Foundation, University Medical School OWL at Bielefeld, Bielefeld University, Campus Bielefeld-Bethel, Burgsteig 13, 33617 Bielefeld, Germany
| | - Christian Kaltschmidt
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany
- Forschungsverbund BioMedizin Bielefeld, OWL (FBMB e.V.), Maraweg 21, 33617 Bielefeld, Germany
| |
Collapse
|
|
3
|
Gao B, Li X, Li S, Wang S, Wu J, Li J. Pan-cancer analysis identifies RNA helicase DDX1 as a prognostic marker. PHENOMICS (CHAM, SWITZERLAND) 2022; 2:33-49. [PMID: 36939765 PMCID: PMC9590584 DOI: 10.1007/s43657-021-00034-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 10/29/2021] [Accepted: 11/01/2021] [Indexed: 10/19/2022]
Abstract
The DEAD-box RNA helicase (DDX) family plays a critical role in the growth and development of multiple organisms. DDX1 is involved in mRNA/rRNA processing and mature, virus replication and transcription, hormone metabolism, tumorigenesis, and tumor development. However, how DDX1 functions in various cancers remains unclear. Here, we explored the potential oncogenic roles of DDX1 across 33 tumors with The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) databases. DDX1 is highly expressed in breast cancer (BRCA), cholangiocarcinoma (CHOL), and colon adenocarcinoma (COAD), but it is lowly expressed in renal cancers, including kidney renal clear cell carcinoma (KIRC), kidney chromophobe (KICH), and kidney renal papillary cell carcinoma (KIRP). Low expression of DDX1 in KIRC is correlated with a good prognosis of overall survival (OS) and disease-free survival (DFS). Highly expressed DDX1 is linked to a poor prognosis of OS for adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), KICH, and liver hepatocellular carcinoma (LIHC). Also, the residue Ser481 of DDX1 had an enhanced phosphorylation level in BRCA and ovarian cancer (OV) but decreased in KIRC. Immune infiltration analysis exhibited that DDX1 expression affected CD8+ T cells, and it was significantly associated with MSI (microsatellite instability), TMB (tumor mutational burden), and ICT (immune checkpoint blockade therapy) in tumors. In addition, the depletion of DDX1 dramatically affected the cell viability of human tumor-derived cell lines. DDX1 could affect the DNA repair pathway and the RNA transport/DNA replication processes during tumorigenesis by analyzing the CancerSEA database. Thus, our pan-cancer analysis revealed that DDX1 had complicated impacts on different cancers and might act as a prognostic marker for cancers such as renal cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s43657-021-00034-x.
Collapse
Affiliation(s)
- Baocai Gao
- State Key Laboratory of Genetic Engineering, School of Life Sciences, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, 200438 China
| | - Xiangnan Li
- State Key Laboratory of Genetic Engineering, School of Life Sciences, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, 200438 China
| | - Shujie Li
- Kunming Institute of Physics, Kunming, 650223 China
| | - Sen Wang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, 200438 China
| | - Jiaxue Wu
- State Key Laboratory of Genetic Engineering, School of Life Sciences, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, 200438 China
| | - Jixi Li
- State Key Laboratory of Genetic Engineering, School of Life Sciences, MOE Engineering Research Center of Gene Technology, Shanghai Engineering Research Center of Industrial Microorganisms, Fudan University, Shanghai, 200438 China
| |
Collapse
|
|
4
|
High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis. Int J Mol Sci 2021; 22:ijms222212487. [PMID: 34830369 PMCID: PMC8619409 DOI: 10.3390/ijms222212487] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2021] [Revised: 11/10/2021] [Accepted: 11/10/2021] [Indexed: 11/16/2022] Open
Abstract
It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene hmg was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed hmg gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of bar and hmg with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the bar and hmg genes varied from 1.18 to 2.94%. The copy numbers of the transgene bar using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T1 and T2 progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new hmg gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.
Collapse
|
|
5
|
Windmöller BA, Beshay M, Helweg LP, Flottmann C, Beermann M, Förster C, Wilkens L, Greiner JFW, Kaltschmidt C, Kaltschmidt B. Novel Primary Human Cancer Stem-Like Cell Populations from Non-Small Cell Lung Cancer: Inhibition of Cell Survival by Targeting NF-κB and MYC Signaling. Cells 2021; 10:cells10051024. [PMID: 33925297 PMCID: PMC8145874 DOI: 10.3390/cells10051024] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2021] [Revised: 04/21/2021] [Accepted: 04/23/2021] [Indexed: 02/07/2023] Open
Abstract
There is growing evidence that cancer stem cells (CSCs), a small subpopulation of self-renewal cancer cells, are responsible for tumor growth, treatment resistance, and cancer relapse and are thus of enormous clinical interest. Here, we aimed to isolate new CSC-like cells derived from human primary non-small cell lung cancer (NSCLC) specimens and to analyze the influence of different inhibitors of NF-κB and MYC signaling on cell survival. CSC-like cells were established from three squamous cell carcinomas (SCC) and three adenocarcinomas (AC) of the lung and were shown to express common CSC markers such as Prominin-1, CD44-antigen, and Nestin. Further, cells gave rise to spherical cancer organoids. Inhibition of MYC and NF-κB signaling using KJ-Pyr-9, dexamethasone, and pyrrolidinedithiocarbamate resulted in significant reductions in cell survival for SCC- and AC-derived cells. However, inhibition of the protein–protein interaction of MYC/NMYC proto-oncogenes with Myc-associated factor X (MAX) using KJ-Pyr-9 revealed the most promising survival-decreasing effects. Next to the establishment of six novel in vitro models for studying NSCLC-derived CSC-like populations, the presented investigations might provide new insights into potential novel therapies targeting NF-κB/MYC to improve clinical outcomes in NSCLC patients. Nevertheless, the full picture of downstream signaling still remains elusive.
Collapse
Affiliation(s)
- Beatrice A. Windmöller
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany; (L.P.H.); (C.F.); (M.B.); (J.F.W.G.); (C.K.); (B.K.)
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
- Correspondence: ; Tel.: +49-0521-106-5629
| | - Morris Beshay
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
- Department of General Thoracic Surgery, Protestant Hospital of Bethel Foundation, Burgsteig 13, 33617 Bielefeld, Germany
| | - Laureen P. Helweg
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany; (L.P.H.); (C.F.); (M.B.); (J.F.W.G.); (C.K.); (B.K.)
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
| | - Clara Flottmann
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany; (L.P.H.); (C.F.); (M.B.); (J.F.W.G.); (C.K.); (B.K.)
| | - Miriam Beermann
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany; (L.P.H.); (C.F.); (M.B.); (J.F.W.G.); (C.K.); (B.K.)
| | - Christine Förster
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
- Institute of Pathology, KRH Hospital Nordstadt, Haltenhoffstrasse 41, Affiliated with the Protestant Hospital of Bethel Foundation, 30167 Hannover, Germany
| | - Ludwig Wilkens
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
- Institute of Pathology, KRH Hospital Nordstadt, Haltenhoffstrasse 41, Affiliated with the Protestant Hospital of Bethel Foundation, 30167 Hannover, Germany
| | - Johannes F. W. Greiner
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany; (L.P.H.); (C.F.); (M.B.); (J.F.W.G.); (C.K.); (B.K.)
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
| | - Christian Kaltschmidt
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany; (L.P.H.); (C.F.); (M.B.); (J.F.W.G.); (C.K.); (B.K.)
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
| | - Barbara Kaltschmidt
- Department of Cell Biology, University of Bielefeld, Universitätsstrasse 25, 33615 Bielefeld, Germany; (L.P.H.); (C.F.); (M.B.); (J.F.W.G.); (C.K.); (B.K.)
- Forschungsverbund BioMedizin Bielefeld/OWL FBMB e. V., Maraweg 21, 33617 Bielefeld, Germany; (M.B.); (C.F.); (L.W.)
- Molecular Neurobiology, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany
| |
Collapse
|
|
6
|
Kohl S, Llavona P, Sauer A, Reuter P, Weisschuh N, Kempf M, Dehmelt FA, Arrenberg AB, Sliesoraityte I, Zrenner E, van Schooneveld MJ, Rudolph G, Kühlewein L, Wissinger B. A duplication on chromosome 16q12 affecting the IRXB gene cluster is associated with autosomal dominant cone dystrophy with early tritanopic color vision defect. Hum Mol Genet 2021; 30:1218-1229. [PMID: 33891002 PMCID: PMC8212766 DOI: 10.1093/hmg/ddab117] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Revised: 04/15/2021] [Accepted: 04/15/2021] [Indexed: 02/06/2023] Open
Abstract
Cone dystrophies are a rare subgroup of inherited retinal dystrophies and hallmarked by color vision defects, low or decreasing visual acuity and central vision loss, nystagmus and photophobia. Applying genome-wide linkage analysis and array comparative genome hybridization, we identified a locus for autosomal dominant cone dystrophy on chromosome 16q12 in four independent multigeneration families. The locus is defined by duplications of variable size with a smallest region of overlap of 608 kb affecting the IRXB gene cluster and encompasses the genes IRX5 and IRX6. IRX5 and IRX6 belong to the Iroquois (Iro) protein family of homeodomain-containing transcription factors involved in patterning and regionalization of embryonic tissue in vertebrates, including the eye and the retina. All patients presented with a unique progressive cone dystrophy phenotype hallmarked by early tritanopic color vision defects. We propose that the disease underlies a misregulation of the IRXB gene cluster on chromosome 16q12 and demonstrate that overexpression of Irx5a and Irx6a, the two orthologous genes in zebrafish, results in visual impairment in 5-day-old zebrafish larvae.
Collapse
Affiliation(s)
- Susanne Kohl
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany
| | - Pablo Llavona
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany
| | - Alexandra Sauer
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany
| | - Peggy Reuter
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany
| | - Nicole Weisschuh
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany
| | - Melanie Kempf
- University Eye Hospital, Centre for Ophthalmology, University of Tübingen, Universitätsklinikum Tübingen, Tübingen 72076, Germany.,Center for Rare Eye Diseases, University of Tübingen, Tübingen 72076, Germany
| | - Florian Alexander Dehmelt
- Werner Reichardt Centre for Integrative Neuroscience and Institute of Neurobiology, University of Tübingen, Tübingen 72076, Germany
| | - Aristides B Arrenberg
- Werner Reichardt Centre for Integrative Neuroscience and Institute of Neurobiology, University of Tübingen, Tübingen 72076, Germany
| | - Ieva Sliesoraityte
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany
| | - Eberhart Zrenner
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany.,Werner Reichardt Centre for Integrative Neuroscience and Institute of Neurobiology, University of Tübingen, Tübingen 72076, Germany
| | - Mary J van Schooneveld
- Department of Ophthalmology, Amsterdam University Medical Centre, Amsterdam 1100 DD, The Netherlands.,Bartiméus Diagnostic Department, Zeist, The Netherlands
| | - Günther Rudolph
- Department of Ophthalmology, University Hospital, LMU Munich, München 80336, Germany
| | - Laura Kühlewein
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany.,University Eye Hospital, Centre for Ophthalmology, University of Tübingen, Universitätsklinikum Tübingen, Tübingen 72076, Germany
| | - Bernd Wissinger
- Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen 72076, Germany
| |
Collapse
|
|
7
|
Schulte am Esch J, Windmöller BA, Hanewinkel J, Storm J, Förster C, Wilkens L, Krüger M, Kaltschmidt B, Kaltschmidt C. Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition. Cancers (Basel) 2020; 12:cancers12092582. [PMID: 32927768 PMCID: PMC7564713 DOI: 10.3390/cancers12092582] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 09/04/2020] [Accepted: 09/08/2020] [Indexed: 12/24/2022] Open
Abstract
Simple Summary The aim of this study was to gain a better understanding of cancer stem cells, which are a small subpopulation of tumor cells with high plasticity driving tumor growth and metastasis. Here we isolated two novel colorectal cancer cell lines originating from a rectal neuroendocrine carcinoma and a colorectal adenocarcinoma, depicting stem-like properties. These in vitro models offer the possibility to evaluate pathophysiological mechanisms in order to develop tailored therapeutic strategies for distinct colorectal malignancies. Investigations revealed gene copy number gain of the N-myc proto-oncogene for both. Accordingly, inhibition of the protein–protein interaction of myc and N-myc proto-oncogenes with the myc-associated factor X utilizing small molecule KJ-Pyr-9, exhibited a significant reduction in survival of both cell lines by the induction of apoptosis. Consequently, the blockage of these interactions may serve as a possible treatment strategy for colorectal cancer cell lines with gene copy number gain of the N-myc proto-oncogene. Abstract Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial–mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.
Collapse
Affiliation(s)
- Jan Schulte am Esch
- Department of General and Visceral Surgery, Protestant Hospital of Bethel Foundation, 33611 Bielefeld, Germany;
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
| | - Beatrice Ariane Windmöller
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
- Correspondence: ; Tel.: +49-0521-106-5629
| | - Johannes Hanewinkel
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
| | - Jonathan Storm
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
| | - Christine Förster
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Institute of Pathology, KRH Hospital Nordstadt, affiliated with the Protestant Hospital of Bethel Foundation, 30167 Hannover, Germany
| | - Ludwig Wilkens
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Institute of Pathology, KRH Hospital Nordstadt, affiliated with the Protestant Hospital of Bethel Foundation, 30167 Hannover, Germany
| | - Martin Krüger
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Internal Medicine and Gastroenterology, Protestant Hospital of Bethel Foundation, 33611 Bielefeld, Germany
| | - Barbara Kaltschmidt
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
- Molecular Neurobiology, University of Bielefeld, 33615 Bielefeld, Germany
| | - Christian Kaltschmidt
- Forschungsverbund BioMedizin Bielefeld (FBMB), 33611 Bielefeld, Germany; (J.S.); (C.F.); (L.W.); (M.K.); (B.K.); (C.K.)
- Department of Cell Biology, University of Bielefeld, 33611 Bielefeld, Germany;
| |
Collapse
|
|
8
|
Felden J, Baumann B, Ali M, Audo I, Ayuso C, Bocquet B, Casteels I, Garcia-Sandoval B, Jacobson SG, Jurklies B, Kellner U, Kessel L, Lorenz B, McKibbin M, Meunier I, de Ravel T, Rosenberg T, Rüther K, Vadala M, Wissinger B, Stingl K, Kohl S. Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene. Hum Mutat 2019; 40:1145-1155. [PMID: 31058429 DOI: 10.1002/humu.23768] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2018] [Revised: 04/03/2019] [Accepted: 04/18/2019] [Indexed: 02/04/2023]
Abstract
Achromatopsia (ACHM) is a hereditary cone photoreceptor disorder characterized by the inability to discriminate colors, nystagmus, photophobia, and low-visual acuity. Six genes have been associated with this rare autosomal recessively inherited disease, including the GNAT2 gene encoding the catalytic α-subunit of the G-protein transducin which is expressed in the cone photoreceptor outer segment. Out of a cohort of 1,116 independent families diagnosed with a primary clinical diagnosis of ACHM, we identified 23 patients with ACHM from 19 independent families with likely causative mutations in GNAT2, representing 1.7% of our large ACHM cohort. In total 22 different potentially disease-causing variants, of which 12 are novel, were identified. The mutation spectrum also includes a novel copy number variation, a heterozygous duplication of exon 4, of which the breakpoint matches exactly that of the previously reported exon 4 deletion. Two patients carry just a single heterozygous variant. In addition to our previous study on GNAT2-ACHM, we also present detailed clinical data of these patients.
Collapse
Affiliation(s)
- Julia Felden
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Tuebingen, Tuebingen, Germany
| | - Britta Baumann
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Tuebingen, Tuebingen, Germany
| | - Manir Ali
- Section of Ophthalmology and Neuroscience, Leeds Institute of Medical Research at St. James's University Hospital, University of Leeds, Leeds, England
| | - Isabelle Audo
- Sorbonne Universités, UPMC Univ Paris 06, INSERM, CNRS, Institute de la Vision/ CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DHOS, Paris, France
| | - Carmen Ayuso
- University Hospital Fundación Jiménez Díaz/Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain
| | - Beatrice Bocquet
- Centre de Référence Maladies Sensorielles Génétiques, Hôpital Gui de Chauliac; Montpellier University and INSERM U1051, Institute for Neurosciences of Montpellier, Montpellier, France
| | - Ingele Casteels
- Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium
| | | | - Samuel G Jacobson
- Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | | | - Ulrich Kellner
- Rare Retinal Disease Center, AugenZentrum Siegburg, MVZ ADTC Siegburg GmbH, Europaplatz 3, Siegburg, Germany
| | - Line Kessel
- The National Eye Clinic, Rigshospitalet, Kennedy Center, Glostrup, Denmark.,Department of Clinical Medicine, University of Copenhagen, Denmark
| | - Birgit Lorenz
- Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany
| | - Martin McKibbin
- Section of Ophthalmology and Neuroscience, Leeds Institute of Medical Research at St. James's University Hospital, University of Leeds, Leeds, England
| | - Isabelle Meunier
- Centre de Référence Maladies Sensorielles Génétiques, Hôpital Gui de Chauliac; Montpellier University and INSERM U1051, Institute for Neurosciences of Montpellier, Montpellier, France
| | - Thomy de Ravel
- Center for Human Genetics, University Hospitals Leuven, University of Leuven, Leuven, Belgium
| | - Thomas Rosenberg
- The National Eye Clinic, Rigshospitalet, Kennedy Center, Glostrup, Denmark.,Department of Clinical Medicine, University of Copenhagen, Denmark
| | - Klaus Rüther
- Augenarztpraxis, Dorotheenstrasse 56, Berlin, Germany
| | - Maria Vadala
- Ophthalmology Institute, Dipartimento di Biomedicina, Neuroscienze e Diagnostica Avanzata (BiND), Università degli Studi di Palermo
| | - Bernd Wissinger
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Tuebingen, Tuebingen, Germany
| | - Katarina Stingl
- University Eye Hospital, Center for Ophthalmology, University of Tübingen, Germany
| | - Susanne Kohl
- Institute for Ophthalmic Research, Centre for Ophthalmology, University Tuebingen, Tuebingen, Germany
| |
Collapse
|
|
9
|
Iehara T, Yagyu S, Gotoh T, Ouchi K, Yoshida H, Miyachi M, Kikuchi K, Sugimoto T, Hosoi H. A prospective evaluation of liquid biopsy for detecting MYCN amplification in neuroblastoma patients. Jpn J Clin Oncol 2019; 49:743-748. [DOI: 10.1093/jjco/hyz063] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2019] [Revised: 02/25/2019] [Indexed: 02/06/2023] Open
Abstract
Abstract
Background
Our previous study reported a method for determining MYCN gene amplification (MNA) status using cell-free DNA in serum. We prospectively analyzed the serum MNA status using sera obtained before the initial diagnosis from patients with neuroblastoma and evaluated the utility of this method.
Methods
Eighty patients were enrolled in the study. The serum MYCN/NAGK ratio was assessed for all cases.
Results
Fifteen cases showed serum MNA, while 65 did not. Of the 80 total patients, tumor samples for a genetic analysis were not obtained from 27 due to the patients’ condition or other reasons. For the 43 of 80 cases that had both serum and tumor samples analyzed, the serum-based MNA status matched to tumor-based MNA status (P < 0.001). The sensitivity and the specificity were 100%, respectively. Seven of 15 cases who diagnosed as MNA by serum-based MNA status were <18 months of age, and tumor samples were not obtained from 4 of these cases. Based on the serum MNA status, these cases were able to start treatment immediately. The 4-year event-free survival rates of cases with and without MNA in sera were 37.5% and 84.8%, respectively (P < 0.001).
Conclusion
The serum-based MNA status was useful for precisely predicting the MNA status in tumor and it has clinical benefits for predicting risk stratification in patients for whom obtaining tumor samples is difficult.
Collapse
Affiliation(s)
- Tomoko Iehara
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Shigeki Yagyu
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Takahiro Gotoh
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Kazutaka Ouchi
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Hideki Yoshida
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Mitsuru Miyachi
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Ken Kikuchi
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Tohru Sugimoto
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| | - Hajime Hosoi
- Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto 602-8566, Japan
| |
Collapse
|
|
10
|
Salgado-Freiría R, López-Doval S, Lafuente A. Perfluorooctane sulfonate (PFOS) can alter the hypothalamic–pituitary–adrenal (HPA) axis activity by modifying CRF1 and glucocorticoid receptors. Toxicol Lett 2018; 295:1-9. [DOI: 10.1016/j.toxlet.2018.05.025] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2017] [Revised: 03/28/2018] [Accepted: 05/20/2018] [Indexed: 12/22/2022]
|
|
11
|
Hélias-Rodzewicz Z, Lourenco N, Bakari M, Capron C, Emile JF. CDKN2A Depletion Causes Aneuploidy and Enhances Cell Proliferation in Non-Immortalized Normal Human Cells. Cancer Invest 2018; 36:338-348. [PMID: 30136875 DOI: 10.1080/07357907.2018.1491588] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Aneuploidy is a common feature of cancer cells and may contribute to cellular transformation and cancer development. In this study, we found that significant down-regulation of CDKN2A, CHEK2, CDCA8, TP53BP1, and CCNDBP1 led to chromosome imbalances in two diploid non-immortalized human cell lines; however, only CDKN2A inhibition enhanced cell proliferation and additionally up-regulated three cell cycle control genes: CDCA8, AURKA, and CCND. These results confirm that CDKN2A is a tumor suppressor gene driving human cancer development by inducing cell aneuploidy and cell cycle up-regulation.
Collapse
Affiliation(s)
- Zofia Hélias-Rodzewicz
- a EA4340, UVSQ , Boulogne-Billancourt , France.,b Service de Pathologie, CHU Ambroise Paré , Boulogne-Billancourt , France
| | - Nelson Lourenco
- a EA4340, UVSQ , Boulogne-Billancourt , France.,c Service de Gastroenterologie, Hopital St Louis, APHP , Paris, France
| | | | - Claude Capron
- a EA4340, UVSQ , Boulogne-Billancourt , France.,d Service de Hématologie-Immunologie, CHU Ambroise Paré , Boulogne-Billancourt , France
| | - Jean-François Emile
- a EA4340, UVSQ , Boulogne-Billancourt , France.,b Service de Pathologie, CHU Ambroise Paré , Boulogne-Billancourt , France
| |
Collapse
|
|
12
|
Tokunaga R, Imamura Y, Nakamura K, Ishimoto T, Nakagawa S, Miyake K, Nakaji Y, Tsuda Y, Iwatsuki M, Baba Y, Sakamoto Y, Miyamoto Y, Saeki H, Yoshida N, Oki E, Watanabe M, Oda Y, Bass AJ, Maehara Y, Baba H. Fibroblast growth factor receptor 2 expression, but not its genetic amplification, is associated with tumor growth and worse survival in esophagogastric junction adenocarcinoma. Oncotarget 2017; 7:19748-61. [PMID: 26933914 PMCID: PMC4991416 DOI: 10.18632/oncotarget.7782] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2015] [Accepted: 01/31/2016] [Indexed: 12/12/2022] Open
Abstract
Background Fibroblast growth factor receptor 2 (FGFR2) genetic alterations lead to tumor cell proliferation in various types of cancer. We hypothesized that FGFR2 amplification is associated with FGFR2 expression, resulting in tumor growth and poorer outcome in esophagogastric junction (EGJ) adenocarcinoma. Patients and Methods A total of 176 consecutive chemo-naive patients with EGJ adenocarcinoma were enrolled from two academic institutions. FGFR2 amplification was examined by real-time PCR (N = 140) and FGFR2 expression with immunohistochemical staining (N = 176), and compared against clinicopathological factors and patient outcomes. The effects of FGFR2 inhibition or overexpression on cell proliferation, cell cycle, and apoptosis assays were investigated in EGJ adenocarcinoma cell lines. Downstream FGFR2, AKT and ERK were also examined. Results Based on the correlation between FGFR2 levels and FGFR2 overexpression in vitro, FGFR2 amplification was defined as copy number > 3.0. In clinical samples, FGFR2 amplification and FGFR2 IHC expression were 15% and 61%, respectively. Although these two statuses were significantly correlated (P < 0.05), only FGFR2 IHC expression was significantly associated with tumor depth (multivariate P < 0.001) and overall survival of patients (univariate P = 0.007). Supporting these findings, FGFR2 overexpression was associated with tumor cell proliferation, cell cycle progression, and anti-apoptosis. Selective inhibition of FGFR2 sufficiently suppressed tumor cell proliferation through de-phosphorylation of AKT and ERK. Conclusions FGFR2 amplification was significantly associated with FGFR2 expression. FGFR2 expression (but not FGFR2 amplification) was associated with tumor growth and patient outcomes. Our findings support FGFR2 as a novel therapeutic target for EGJ adenocarcinoma.
Collapse
Affiliation(s)
- Ryuma Tokunaga
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Yu Imamura
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.,Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Kenichi Nakamura
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Takatsugu Ishimoto
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Shigeki Nakagawa
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Keisuke Miyake
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Yu Nakaji
- Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.,Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Yasuo Tsuda
- Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Masaaki Iwatsuki
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Yoshifumi Baba
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Yasuo Sakamoto
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Yuji Miyamoto
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Hiroshi Saeki
- Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Naoya Yoshida
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| | - Eiji Oki
- Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Masayuki Watanabe
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.,Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Yoshinao Oda
- Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Adam J Bass
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.,Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.,Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA
| | - Yoshihiko Maehara
- Department of Surgery and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Hideo Baba
- Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
| |
Collapse
|
|
13
|
Yang L, Wang YZ, Zhu HH, Chang Y, Li LD, Chen WM, Long LY, Zhang YH, Liu YR, Lu J, Qin YZ. PRAME Gene Copy Number Variation Is Related to Its Expression in Multiple Myeloma. DNA Cell Biol 2017; 36:1099-1107. [PMID: 28953414 DOI: 10.1089/dna.2017.3951] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Multiple myeloma (MM) patients commonly present abnormal expression of cancer-testis antigens, which may serve as immunotherapeutic targets and prognostic factors. We previously reported that preferentially expressed antigen of melanoma (PRAME) overexpression in bone marrow mononuclear cells is related to progression in MM patients treated with non-bortezomib-containing regimens. The mechanism underlying variations in PRAME expression remains unknown. To investigate the impact of gene copy number variation (CNV) on PRAME expression, plasma cells were sorted from 50 newly diagnosed patients and 8 healthy volunteers to measure PRAME transcript levels and gene copy numbers by real-time quantitative polymerase chain reaction. A total of 14 (28.0%), 7 (14.0%), and 29 (58.0%) patients exhibited overexpression, expression within the normal range, and low expression, respectively. PRAME overexpression was significantly related to a lower 1-year progression-free survival rate compared with PRAME low expression (20.0% vs. 88.9%, p = 0.043). The mean PRAME gene copy number relative to albumin (ALB) in normal samples was ∼1.0, whereas 4.0%, 24.0%, 70.0%, and 2.0% of patients had PRAME gene relative copy numbers of approximately 0, 0.5, 1.0, and 2.0, respectively. Patients with PRAME gene deletion (relative copy number of 0 or 0.5) had significantly higher frequency of PRAME nonoverexpression and lambda light chain expression than those with no deletion (p = 0.011 and 0.003). Thus, PRAME gene CNV occurs in MM. Gene deletion may be one mechanism leading to PRAME nonoverexpression and related to immunoglobulin lambda light chain locus rearrangement. PRAME overexpression in plasma cells might be an adverse prognostic factor for progression in MM.
Collapse
Affiliation(s)
- Lu Yang
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Ya-Zhe Wang
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Hong-Hu Zhu
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Yan Chang
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Ling-Di Li
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Wen-Min Chen
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Ling-Yu Long
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Yan-Huan Zhang
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Yan-Rong Liu
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Jin Lu
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| | - Ya-Zhen Qin
- Peking University People's Hospital, Peking University Institute of Hematology , Beijing, China
| |
Collapse
|
|
14
|
Linares-Clemente P, Aguilar-Morante D, Rodríguez-Prieto I, Ramírez G, de Torres C, Santamaría V, Pascual-Vaca D, Colmenero-Repiso A, Vega FM, Mora J, Cabello R, Márquez C, Rivas E, Pardal R. Neural crest derived progenitor cells contribute to tumor stroma and aggressiveness in stage 4/M neuroblastoma. Oncotarget 2017; 8:89775-89792. [PMID: 29163787 PMCID: PMC5685708 DOI: 10.18632/oncotarget.21128] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2016] [Accepted: 09/04/2017] [Indexed: 12/15/2022] Open
Abstract
Pediatric tumors arise upon oncogenic transformation of stem/progenitor cells during embryonic development. Given this scenario, the existence of non-tumorigenic stem cells included within the aberrant tumoral niche, with a potential role in tumor biology, is an intriguing and unstudied possibility. Here, we describe the presence and function of non-tumorigenic neural crest-derived progenitor cells in aggressive neuroblastoma (NB) tumors. These cells differentiate into neural crest typical mesectodermal derivatives, giving rise to tumor stroma and promoting proliferation and tumor aggressiveness. Furthermore, an analysis of gene expression profiles in stage 4/M NB revealed a neural crest stem cell (NCSC) gene signature that was associated to stromal phenotype and high probability of relapse. Thus, this NCSC gene expression signature could be used in prognosis to improve stratification of stage 4/M NB tumors. Our results might facilitate the design of new therapies by targeting NCSCs and their contribution to tumor stroma.
Collapse
Affiliation(s)
- Pedro Linares-Clemente
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain
| | - Diana Aguilar-Morante
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain
| | - Ismael Rodríguez-Prieto
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain
| | - Gema Ramírez
- Departamento de Oncología Pediátrica, Hospital Universitario Virgen del Rocío, Sevilla, Spain
| | - Carmen de Torres
- Departamento de Oncología, Hospital Sant Joan de Déu, Barcelona, Spain
| | - Vicente Santamaría
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain.,Departamento de Oncología Pediátrica, Hospital Universitario Virgen del Rocío, Sevilla, Spain
| | - Diego Pascual-Vaca
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain.,Departamento de Anatomía Patológica, Hospital Universitario Virgen del Rocío, Sevilla, Spain
| | - Ana Colmenero-Repiso
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain
| | - Francisco M Vega
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain
| | - Jaume Mora
- Departamento de Oncología, Hospital Sant Joan de Déu, Barcelona, Spain
| | - Rosa Cabello
- Departamento de Cirugía Pediátrica, Hospital Universitario Virgen del Rocío, Sevilla, Spain
| | - Catalina Márquez
- Departamento de Oncología Pediátrica, Hospital Universitario Virgen del Rocío, Sevilla, Spain
| | - Eloy Rivas
- Departamento de Anatomía Patológica, Hospital Universitario Virgen del Rocío, Sevilla, Spain
| | - Ricardo Pardal
- Instituto de Biomedicina de Sevilla (IBiS), Departamento de Fisiología Médica y Biofísica, Hospital Universitario Virgen del Rocío, CSIC, Universidad de Sevilla, Sevilla, Spain
| |
Collapse
|
|
15
|
Chillón MC, Jiménez C, García-Sanz R, Alcoceba M, Prieto I, García-Alvarez M, Antón A, Maldonado R, Hernández-Ruano M, González M, Gutiérrez NC, Sarasquete ME. Quantitative PCR: an alternative approach to detect common copy number alterations in multiple myeloma. Ann Hematol 2017; 96:1699-1705. [PMID: 28770277 DOI: 10.1007/s00277-017-3083-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2017] [Accepted: 07/25/2017] [Indexed: 12/25/2022]
Abstract
Chromosome 1q gains and 13q deletions are common cytogenetic aberrations in multiple myeloma (MM) that confer a poor prognosis. There are several techniques for the targeted study of these alterations, but interphase fluorescence in situ hybridization (FISH) is the current gold standard. The aim of the present study was to validate quantitative PCR (qPCR) as an alternative to FISH studies in CD138+-enriched plasma cells (PCs) from MM patients at diagnosis. We analyzed 1q gains and 13q deletions by qPCR in 57 and 60 MM patients, respectively. qPCR applicability was 84 and 88% for 1q and 13q, respectively. The qPCR and FISH methods had a sensitivity and specificity of 88 and 71% for 1q gains, and 79 and 100% for 13q deletions. A second qPCR assay for each region was carried out to confirm the previous results. Paired qPCR (two assays) and FISH results were available from 53 MM patients: 26 for 1q amplification and 27 for 13q deletion. qPCR assays gave concordant results (qPCR-consistent) in 20 of the 26 (77%) 1q gains and 25 of the 27 (93%) 13q deletions. Considering only the consistent data, the overall concordance among qPCR and FISH was 85 and 100% for 1q gains and 13q deletions, respectively. Our results show a substantial agreement between qPCR and the gold standard FISH technique, indicating the potential of qPCR as an alternative approach, particularly when the starting material is too scarce or cells are too damaged to obtain accurate results from FISH studies.
Collapse
Affiliation(s)
- M C Chillón
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain.,CIBERONC, Madrid, Spain
| | - C Jiménez
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain
| | - R García-Sanz
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain. .,CIBERONC, Madrid, Spain.
| | - M Alcoceba
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain.,CIBERONC, Madrid, Spain
| | - I Prieto
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain
| | - M García-Alvarez
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain
| | - A Antón
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain
| | - R Maldonado
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain
| | - M Hernández-Ruano
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain
| | - M González
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain.,CIBERONC, Madrid, Spain
| | - N C Gutiérrez
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain
| | - M E Sarasquete
- Hospital Universitario de Salamanca-Instituto de Investigación Biomédica de Salamanca (IBSAL), Centro de Investigación del Cáncer-IBMCC-CSIC, Paseo de San Vicente 58-182, 37007, Salamanca, Spain.,CIBERONC, Madrid, Spain
| |
Collapse
|
|
16
|
Gnanamony M, Antony R, Fernández KS, Jaime L, Lin J, Joseph PA, Gondi CS. Chronic radiation exposure of neuroblastoma cells reduces nMYC copy number. Oncol Lett 2017; 14:3363-3370. [PMID: 28927089 PMCID: PMC5587969 DOI: 10.3892/ol.2017.6652] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Accepted: 03/03/2017] [Indexed: 12/02/2022] Open
Abstract
Neuroblastoma accounts for >15% of cancer-associated mortalities of children in the USA. Despite aggressive treatment regimens, the long-term survival for these children remains <40%. The identification of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (nMYC) gene amplification during diagnosis is associated with poor prognosis in neuroblastoma. There are limited studies examining changes in nMYC copy numbers in response to therapy and its biological effect on cancer cells. The aim of the present study was to evaluate the effect of radiation on nMYC expression and amplification status in high-risk neuroblastoma. The effect of acute (5 Gy) and chronic (25 Gy) radiation on two nMYC-amplified cell lines, SK-N-BE (2) and NB-1691, was investigated. The results demonstrate that, following chronic but not acute radiation, the two cell lines regained their proliferation potential similar to the controls. This increased proliferation was characterized by loss of nMYC mRNA and protein expression. It was also revealed that nMYC loss was accompanied by nuclear localization of c-Myc. Using fluorescent in situ hybridization and quantitative polymerase chain reaction analysis, the results of the present study demonstrated that chronic radiation causes a severe loss of nMYC gene copy number. The present study is the first to provide experimental evidence that prolonged radiation therapy affects nMYC gene copy number in high-risk neuroblastoma but does not significantly improve the prognostic outlook.
Collapse
Affiliation(s)
- Manu Gnanamony
- Department of Pediatrics, University of Illinois College of Medicine, Peoria, IL 61605, USA
| | - Reuben Antony
- UC Davis Comprehensive Cancer Center, Pediatric Hematology and Oncology, UC Davis Children's Hospital, Sacramento, CA 95817, USA
| | - Karen S Fernández
- Cancer and Blood Diseases Center, 9300 Valley Children's Place FC13, Madera, CA 93636, USA
| | - Libes Jaime
- Department of Pediatrics, University of Illinois College of Medicine, Peoria, IL 61605, USA
| | - Julian Lin
- Department of Neurosurgery, University of Illinois College of Medicine, Peoria, IL 61605, USA
| | - Pushpa A Joseph
- Department of Pathology, University of Illinois College of Medicine, Peoria, IL 61605, USA
| | - Christopher S Gondi
- Department of Pathology, University of Illinois College of Medicine, Peoria, IL 61605, USA.,Department of Internal Medicine, University of Illinois College of Medicine, Peoria, IL 61605, USA.,Department of Surgery, University of Illinois College of Medicine, Peoria, IL 61605, USA
| |
Collapse
|
|
17
|
Van Roy N, Van Der Linden M, Menten B, Dheedene A, Vandeputte C, Van Dorpe J, Laureys G, Renard M, Sante T, Lammens T, De Wilde B, Speleman F, De Preter K. Shallow Whole Genome Sequencing on Circulating Cell-Free DNA Allows Reliable Noninvasive Copy-Number Profiling in Neuroblastoma Patients. Clin Cancer Res 2017; 23:6305-6314. [PMID: 28710315 DOI: 10.1158/1078-0432.ccr-17-0675] [Citation(s) in RCA: 90] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2017] [Revised: 06/02/2017] [Accepted: 07/10/2017] [Indexed: 11/16/2022]
Abstract
Purpose: Neuroblastoma (NB) is a heterogeneous disease characterized by distinct clinical features and by the presence of typical copy-number alterations (CNAs). Given the strong association of these CNA profiles with prognosis, analysis of the CNA profile at diagnosis is mandatory. Therefore, we tested whether the analysis of circulating cell-free DNA (cfDNA) present in plasma samples of patients with NB could offer a valuable alternative to primary tumor DNA for CNA profiling.Experimental Design: In 37 patients with NB, cfDNA analysis using shallow whole genome sequencing (sWGS) was compared with arrayCGH analysis of primary tumor tissue.Results: Comparison of CNA profiles on cfDNA showed highly concordant patterns, particularly in high-stage patients. Numerical chromosome imbalances as well as large and focal structural aberrations including MYCN and LIN28B amplification and ATRX deletion could be readily detected with sWGS using a low input of cfDNA.Conclusions: In conclusion, sWGS analysis on cfDNA offers a cost-effective, noninvasive, rapid, robust and sensitive alternative for tumor DNA copy-number profiling in most patients with NB. Clin Cancer Res; 23(20); 6305-14. ©2017 AACR.
Collapse
Affiliation(s)
- Nadine Van Roy
- Center for Medical Genetics, Ghent University, Ghent, Belgium.,Cancer Research Institute Ghent, Ghent University, Ghent, Belgium
| | - Malaïka Van Der Linden
- Center for Medical Genetics, Ghent University, Ghent, Belgium.,Department of Pathology, Ghent University, Ghent, Belgium
| | - Björn Menten
- Center for Medical Genetics, Ghent University, Ghent, Belgium
| | | | - Charlotte Vandeputte
- Center for Medical Genetics, Ghent University, Ghent, Belgium.,Cancer Research Institute Ghent, Ghent University, Ghent, Belgium
| | - Jo Van Dorpe
- Department of Pathology, Ghent University, Ghent, Belgium
| | - Geneviève Laureys
- Cancer Research Institute Ghent, Ghent University, Ghent, Belgium.,Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
| | - Marleen Renard
- Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Leuven University Hospital, Leuven, Belgium
| | - Tom Sante
- Center for Medical Genetics, Ghent University, Ghent, Belgium
| | - Tim Lammens
- Cancer Research Institute Ghent, Ghent University, Ghent, Belgium.,Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
| | - Bram De Wilde
- Cancer Research Institute Ghent, Ghent University, Ghent, Belgium.,Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium
| | - Frank Speleman
- Center for Medical Genetics, Ghent University, Ghent, Belgium.,Cancer Research Institute Ghent, Ghent University, Ghent, Belgium
| | - Katleen De Preter
- Center for Medical Genetics, Ghent University, Ghent, Belgium. .,Cancer Research Institute Ghent, Ghent University, Ghent, Belgium
| |
Collapse
|
|
18
|
Hoeber J, König N, Trolle C, Lekholm E, Zhou C, Pankratova S, Åkesson E, Fredriksson R, Aldskogius H, Kozlova EN. A Combinatorial Approach to Induce Sensory Axon Regeneration into the Dorsal Root Avulsed Spinal Cord. Stem Cells Dev 2017; 26:1065-1077. [PMID: 28562227 DOI: 10.1089/scd.2017.0019] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Spinal root injuries result in newly formed glial scar formation, which prevents regeneration of sensory axons causing permanent sensory loss. Previous studies showed that delivery of trophic factors or implantation of human neural progenitor cells supports sensory axon regeneration and partly restores sensory functions. In this study, we elucidate mechanisms underlying stem cell-mediated ingrowth of sensory axons after dorsal root avulsion (DRA). We show that human spinal cord neural stem/progenitor cells (hscNSPC), and also, mesoporous silica particles loaded with growth factor mimetics (MesoMIM), supported sensory axon regeneration. However, when hscNSPC and MesoMIM were combined, sensory axon regeneration failed. Morphological and tracing analysis showed that sensory axons grow through the newly established glial scar along "bridges" formed by migrating stem cells. Coimplantation of MesoMIM prevented stem cell migration, "bridges" were not formed, and sensory axons failed to enter the spinal cord. MesoMIM applied alone supported sensory axons ingrowth, but without affecting glial scar formation. In vitro, the presence of MesoMIM significantly impaired migration of hscNSPC without affecting their level of differentiation. Our data show that (1) the ability of stem cells to migrate into the spinal cord and organize cellular "bridges" in the newly formed interface is crucial for successful sensory axon regeneration, (2) trophic factor mimetics delivered by mesoporous silica may be a convenient alternative way to induce sensory axon regeneration, and (3) a combinatorial approach of individually beneficial components is not necessarily additive, but can be counterproductive for axonal growth.
Collapse
Affiliation(s)
- Jan Hoeber
- 1 Department of Neuroscience, Uppsala University , Uppsala, Sweden
| | - Niclas König
- 1 Department of Neuroscience, Uppsala University , Uppsala, Sweden
| | - Carl Trolle
- 1 Department of Neuroscience, Uppsala University , Uppsala, Sweden
| | - Emilia Lekholm
- 1 Department of Neuroscience, Uppsala University , Uppsala, Sweden .,2 Department of Pharmaceutical Biosciences, Uppsala University , Uppsala, Sweden
| | | | - Stanislava Pankratova
- 4 Institute of Neuroscience and Pharmacology, University of Copenhagen , Copenhagen, Denmark
| | - Elisabet Åkesson
- 5 Department of Neurobiology, Care Sciences and Society, Karolinska Institutet , Stockholm, Sweden
| | - Robert Fredriksson
- 2 Department of Pharmaceutical Biosciences, Uppsala University , Uppsala, Sweden
| | - Håkan Aldskogius
- 1 Department of Neuroscience, Uppsala University , Uppsala, Sweden
| | - Elena N Kozlova
- 1 Department of Neuroscience, Uppsala University , Uppsala, Sweden
| |
Collapse
|
|
19
|
Hellsten SV, Eriksson MM, Lekholm E, Arapi V, Perland E, Fredriksson R. The gene expression of the neuronal protein, SLC38A9, changes in mouse brain after in vivo starvation and high-fat diet. PLoS One 2017; 12:e0172917. [PMID: 28235079 PMCID: PMC5325605 DOI: 10.1371/journal.pone.0172917] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2016] [Accepted: 02/03/2017] [Indexed: 11/18/2022] Open
Abstract
SLC38A9 is characterized as a lysosomal component of the amino acid sensing Ragulator-RAG GTPase complex, controlling the mechanistic target of rapamycin complex 1 (mTORC1). Here, immunohistochemistry was used to map SLC38A9 in mouse brain and staining was detected throughout the brain, in cortex, hypothalamus, thalamus, hippocampus, brainstem and cerebellum. More specifically, immunostaining was found in areas known to be involved in amino acid sensing and signaling pathways e.g. piriform cortex and hypothalamus. SLC38A9 immunoreactivity co-localized with both GABAergic and glutamatergic neurons, but not with astrocytes. SLC38A9 play a key role in the mTORC1 pathway, and therefore we performed in vivo starvation and high-fat diet studies, to measure gene expression alterations in specific brain tissues and in larger brain regions. Following starvation, Slc38a9 was upregulated in brainstem and cortex, and in anterior parts of the brain (Bregma 3.2 to -2.1mm). After high-fat diet, Slc38a9 was specifically upregulated in hypothalamus, while overall downregulation was noticed throughout the brain (Bregma 3.2 to -8.6mm).
Collapse
Affiliation(s)
- Sofie V. Hellsten
- Department of Pharmaceutical Bioscience, Molecular Neuropharmacology, Uppsala University, Uppsala SE, Sweden
- * E-mail:
| | - Mikaela M. Eriksson
- Department of Pharmaceutical Bioscience, Molecular Neuropharmacology, Uppsala University, Uppsala SE, Sweden
| | - Emilia Lekholm
- Department of Pharmaceutical Bioscience, Molecular Neuropharmacology, Uppsala University, Uppsala SE, Sweden
| | - Vasiliki Arapi
- Department of Pharmaceutical Bioscience, Molecular Neuropharmacology, Uppsala University, Uppsala SE, Sweden
| | - Emelie Perland
- Department of Pharmaceutical Bioscience, Molecular Neuropharmacology, Uppsala University, Uppsala SE, Sweden
| | - Robert Fredriksson
- Department of Pharmaceutical Bioscience, Molecular Neuropharmacology, Uppsala University, Uppsala SE, Sweden
| |
Collapse
|
|
20
|
Estiarte N, Lawrence C, Sanchis V, Ramos A, Crespo-Sempere A. LaeA and VeA are involved in growth morphology, asexual development, and mycotoxin production in Alternaria alternata. Int J Food Microbiol 2016; 238:153-164. [DOI: 10.1016/j.ijfoodmicro.2016.09.003] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Revised: 07/29/2016] [Accepted: 09/05/2016] [Indexed: 12/21/2022]
|
|
21
|
Delespaul L, Lesluyes T, Pérot G, Brulard C, Lartigue L, Baud J, Lagarde P, Le Guellec S, Neuville A, Terrier P, Vince-Ranchère D, Schmidt S, Debant A, Coindre JM, Chibon F. Recurrent TRIO Fusion in Nontranslocation–Related Sarcomas. Clin Cancer Res 2016; 23:857-867. [DOI: 10.1158/1078-0432.ccr-16-0290] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Revised: 06/27/2016] [Accepted: 07/27/2016] [Indexed: 11/16/2022]
|
|
22
|
López-Doval S, Salgado R, Lafuente A. The expression of several reproductive hormone receptors can be modified by perfluorooctane sulfonate (PFOS) in adult male rats. CHEMOSPHERE 2016; 155:488-497. [PMID: 27151425 DOI: 10.1016/j.chemosphere.2016.04.081] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/18/2015] [Revised: 04/02/2016] [Accepted: 04/20/2016] [Indexed: 06/05/2023]
Abstract
This study was undertaken to evaluate the possible role of several reproductive hormone receptors on the disruption of the hypothalamic-pituitary-testis (HPT) axis activity induced by perfluorooctane sulfonate (PFOS). The studied receptors are the gonadotropin-releasing hormone receptor (GnRHr), luteinizing hormone receptor (LHr), follicle-stimulating hormone receptor (FSHr), and the androgen receptor (Ar). Adult male rats were orally treated with 1.0; 3.0 and 6.0 mg of PFOS kg(-1) d(-1) for 28 days. In general terms, PFOS can modify the relative gene and protein expressions of these receptors in several tissues of the reproductive axis. At the testicular level, apart from the expected inhibition of both gene and protein expressions of FSHr and Ar, PFOS also stimulates the GnRHr protein and the LHr gene expression. The receptors of the main hormones involved in the HPT axis may have an important role in the disruption exerted by PFOS on this axis.
Collapse
MESH Headings
- Alkanesulfonic Acids/chemistry
- Alkanesulfonic Acids/pharmacology
- Animals
- Blotting, Western
- Fluorocarbons/chemistry
- Fluorocarbons/pharmacology
- Follicle Stimulating Hormone/metabolism
- Gene Expression Regulation/drug effects
- Gonadotropin-Releasing Hormone/metabolism
- Luteinizing Hormone/metabolism
- Male
- Polymerase Chain Reaction
- Rats
- Rats, Sprague-Dawley
- Receptors, Androgen/genetics
- Receptors, Androgen/metabolism
- Receptors, FSH/genetics
- Receptors, FSH/metabolism
- Receptors, LH/genetics
- Receptors, LH/metabolism
- Receptors, LHRH/genetics
- Receptors, LHRH/metabolism
- Reproduction/drug effects
- Testis/drug effects
- Testis/metabolism
Collapse
Affiliation(s)
- S López-Doval
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas s/n, 32004 Ourense, Spain
| | - R Salgado
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas s/n, 32004 Ourense, Spain
| | - A Lafuente
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas s/n, 32004 Ourense, Spain.
| |
Collapse
|
|
23
|
Yin J, Li Y, Zhao H, Qin Q, Li X, Huang J, Shi Y, Gong S, Liu L, Fu X, Nie S, Wei S. Copy-number variation of MCL1 predicts overall survival of non-small-cell lung cancer in a Southern Chinese population. Cancer Med 2016; 5:2171-9. [PMID: 27264345 PMCID: PMC4898974 DOI: 10.1002/cam4.774] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2016] [Revised: 04/24/2016] [Accepted: 04/26/2016] [Indexed: 12/18/2022] Open
Abstract
BCL2L1 and MCL1 are key anti‐apoptotic genes, and critical for cancer progression. The prognostic values of BCL2L1 and MCL1 copy‐number variations (CNVs) in non‐small‐cell lung cancer (NSCLC) remain largely unknown. Somatic CNVs in BCL2L1 and MCL1 genes were tested in tumor tissues from 516 NSCLC patients in southern China; afterward, survival analyses were conducted with overall survival (OS) as outcome. Additionally, the associations between CNVs and mRNA expression levels were explored using data from 986 NSCLC patients in the Cancer Genome Atlas project. It was found that amplifications of BCL2L1 and MCL1 were associated with unfavorable OS of NSCLC, with adjusted hazards ratio of 1.62 (95% confident interval [CI] = 1.10–2.40; P = 0.015) and 1.39 (95% CI = 1.05–1.84; P = 0.020), respectively. Amplifications of MCL1, but not BCL2L1, were related with higher mRNA expression levels of corresponding gene, compared with non‐amplifications (P = 0.005). Interestingly, after incorporating with MCL1 CNV status, clinical variables (age, sex, TNM stage, and surgical approach) showed an improved discriminatory ability to classify OS (area under curve increased from 72.2% to 74.1%; P = 0.042, DeLong's test). Overall, MCL1 CNV might be a prognostic biomarker for NSCLC, and additional investigations are needed to validate our findings.
Collapse
Affiliation(s)
- Jieyun Yin
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.,Department of Epidemiology and Biostatistics, School of Public Health, Medical College of Soochow University, 199 Ren-ai Road Industrial Park District, Suzhou, China
| | - Yangkai Li
- Department of Thoracic Surgery, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Hao Zhao
- Department of Social Science and Public Health, School of Basic Medical Science, Jiujiang University, No. 17, Lufeng Road, Jiujiang, 332000, China
| | - Qin Qin
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Xiaorong Li
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jiao Huang
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Yun Shi
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Shufang Gong
- Department of Thoracic Surgery, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Li Liu
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Xiangning Fu
- Department of Thoracic Surgery, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Shaofa Nie
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Sheng Wei
- Department of Epidemiology and Biostatistics and MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
| |
Collapse
|
|
24
|
Bamba T, Hasunuma T, Kondo A. Disruption of PHO13 improves ethanol production via the xylose isomerase pathway. AMB Express 2016; 6:4. [PMID: 26769491 PMCID: PMC4713403 DOI: 10.1186/s13568-015-0175-7] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2015] [Accepted: 12/11/2015] [Indexed: 01/08/2023] Open
Abstract
Xylose is the second most abundant sugar in lignocellulosic materials and can be converted to ethanol by recombinant Saccharomyces cerevisiae yeast strains expressing heterologous genes involved in xylose assimilation pathways. Recent research demonstrated that disruption of the alkaline phosphatase gene, PHO13, enhances ethanol production from xylose by a strain expressing the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes; however, the yield of ethanol is poor. In this study, PHO13 was disrupted in a recombinant strain harboring multiple copies of the xylose isomerase (XI) gene derived from Orpinomyces sp., coupled with overexpression of the endogenous xylulokinase (XK) gene and disruption of GRE3, which encodes aldose reductase. The resulting YΔGP/XK/XI strain consumed 2.08 g/L/h of xylose and produced 0.88 g/L/h of volumetric ethanol, for an 86.8 % theoretical ethanol yield, and only YΔGP/XK/XI demonstrated increase in cell concentration. Transcriptome analysis indicated that expression of genes involved in the pentose phosphate pathway (GND1, SOL3, TAL1, RKI1, and TKL1) and TCA cycle and respiratory chain (NDE1, ACO1, ACO2, SDH2, IDH1, IDH2, ATP7, ATP19, SDH4, SDH3, CMC2, and ATP15) was upregulated in the YΔGP/XK/XI strain. And the expression levels of 125 cell cycle genes were changed by deletion of PHO13.
Collapse
|
|
25
|
PKC activation sensitizes basal-like breast cancer cell lines to Smac mimetics. Cell Death Discov 2016; 2:16002. [PMID: 27551497 PMCID: PMC4979953 DOI: 10.1038/cddiscovery.2016.2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2015] [Accepted: 12/16/2015] [Indexed: 01/01/2023] Open
Abstract
There is a need for novel strategies to initiate cancer cell death. One approach is the use of Smac mimetics, which antagonize inhibitor of apoptosis proteins (IAPs). Recent studies have shown that combinations of Smac mimetics such as LBW242 or LCL161 in combination with chemotherapeutic agents increase cancer cell death. Here we show that the protein kinase C (PKC) activator TPA together with the Smac mimetic LBW242 induces cell death in two basal breast cancer cell lines (MDA-MB-468 and BT-549) that are resistant to Smac mimetic as single agent. Ten other LBW242-insensitive cancer cell lines were not influenced by the TPA+LBW242 combination. The TPA+LBW242 effect was suppressed by the PKC inhibitor GF109203X, indicating dependence on PKC enzymatic activity. The PKC effect was mediated via increased synthesis and release of TNFα, which can induce death in the presence of Smac mimetics. The cell death, coinciding with caspase-3 cleavage, was suppressed by caspase inhibition and preceded by the association of RIP1 with caspase-8, as seen in complex II formation. Smac mimetics, but not TPA, induced the non-canonical NF-κB pathway in both MDA-MB-231 and MDA-MB-468 cells. Blocking the canonical NF-κB pathway suppressed TPA induction of TNFα in MDA-MB-468 cells whereas isolated downregulation of either the canonical or non-canonical pathways did not abolish the Smac mimetic induction of the NF-κB driven genes TNFα and BIRC3 in MDA-MB-231 cells although the absolute levels were suppressed. A combined downregulation of the canonical and non-canonical pathways further suppressed TNFα levels and inhibited Smac mimetic-mediated cell death. Our data suggest that in certain basal breast cancer cell lines co-treatment of TPA with a Smac mimetic induces cell death highlighting the potential of using these pathways as molecular targets for basal-like breast cancers.
Collapse
|
|
26
|
Woollard WJ, Kalaivani NP, Jones CL, Roper C, Tung L, Lee JJ, Thomas BR, Tosi I, Ferreira S, Beyers CZ, McKenzie RCT, Butler RM, Lorenc A, Whittaker SJ, Mitchell TJ. Independent Loss of Methylthioadenosine Phosphorylase (MTAP) in Primary Cutaneous T-Cell Lymphoma. J Invest Dermatol 2016; 136:1238-1246. [PMID: 26872600 DOI: 10.1016/j.jid.2016.01.028] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2015] [Revised: 12/07/2015] [Accepted: 01/20/2016] [Indexed: 10/22/2022]
Abstract
Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment.
Collapse
Affiliation(s)
- Wesley J Woollard
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Nithyha P Kalaivani
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Christine L Jones
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Catherine Roper
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Lam Tung
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Jae Jin Lee
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Bjorn R Thomas
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Isabella Tosi
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Silvia Ferreira
- Viapath, Skin Tumour Unit, St John's Institute of Dermatology, Guy's Hospital, London, UK
| | - Carl Z Beyers
- Viapath, Skin Tumour Unit, St John's Institute of Dermatology, Guy's Hospital, London, UK
| | - Robert C T McKenzie
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Rosie M Butler
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Anna Lorenc
- Transformational Bioinformatics, NIHR Research Biomedical Research Center at Guy's and St Thomas' Hospital Foundation Trust and Kings College London, London, UK
| | - Sean J Whittaker
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK
| | - Tracey J Mitchell
- St John's Institute of Dermatology, Division of Genetics and Molecular Medicine, Faculty of Life Sciences & Medicine, King's College London, London, UK.
| |
Collapse
|
|
27
|
Salgado R, López-Doval S, Pereiro N, Lafuente A. Perfluorooctane sulfonate (PFOS) exposure could modify the dopaminergic system in several limbic brain regions. Toxicol Lett 2016; 240:226-35. [DOI: 10.1016/j.toxlet.2015.10.023] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2015] [Revised: 10/22/2015] [Accepted: 10/26/2015] [Indexed: 01/01/2023]
|
|
28
|
Tiwari A, Swamy S, Gopinath KS, Kumar A. Genomic amplification upregulates estrogen-related receptor alpha and its depletion inhibits oral squamous cell carcinoma tumors in vivo. Sci Rep 2015; 5:17621. [PMID: 26639757 PMCID: PMC4671026 DOI: 10.1038/srep17621] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Accepted: 11/03/2015] [Indexed: 12/12/2022] Open
Abstract
The ESRRA gene encodes a transcription factor and regulates several genes, such as WNT11 and OPN, involved in tumorigenesis. It is upregulated in several cancers, including OSCC. We have previously shown that the tumor suppressor miR-125a targets ESRRA, and its downregulation causes upregulation of ESRRA in OSCC. Upregulation of ESRRA in the absence of downregulation of miR-125a in a subset of OSCC samples suggests the involvement of an alternative mechanism. Using TaqMan(®) copy number assay, here we report for the first time that the genomic amplification of ESRRA causes its upregulation in a subset of OSCC samples. Ectopic overexpression of ESRRA led to accelerated cell proliferation, anchorage-independent cell growth and invasion, and inhibited apoptosis. Whereas, knockdown of ESRRA expression by siRNA led to reduced cell proliferation, anchorage-independent cell growth and invasion, and accelerated apoptosis. Furthermore, the delivery of a synthetic biostable ESRRA siRNA to OSCC cells resulted in regression of xenografts in nude mice. Thus, the genomic amplification of ESRRA is another novel mechanism for its upregulation in OSCC. Based on our in vitro and in vivo experiments, we suggest that targeting ESRRA by siRNA could be a novel therapeutic strategy for OSCC and other cancers.
Collapse
Affiliation(s)
- Ankana Tiwari
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
| | - Shivananda Swamy
- Department of Surgery, Bangalore Institute of Oncology, Bangalore 560027, India
| | | | - Arun Kumar
- Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
| |
Collapse
|
|
29
|
Olde Loohuis NFM, Kole K, Glennon JC, Karel P, Van der Borg G, Van Gemert Y, Van den Bosch D, Meinhardt J, Kos A, Shahabipour F, Tiesinga P, van Bokhoven H, Martens GJM, Kaplan BB, Homberg JR, Aschrafi A. Elevated microRNA-181c and microRNA-30d levels in the enlarged amygdala of the valproic acid rat model of autism. Neurobiol Dis 2015; 80:42-53. [PMID: 25986729 DOI: 10.1016/j.nbd.2015.05.006] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2014] [Revised: 04/14/2015] [Accepted: 05/10/2015] [Indexed: 11/17/2022] Open
Abstract
Autism spectrum disorders are severe neurodevelopmental disorders, marked by impairments in reciprocal social interaction, delays in early language and communication, and the presence of restrictive, repetitive and stereotyped behaviors. Accumulating evidence suggests that dysfunction of the amygdala may be partially responsible for the impairment of social behavior that is a hallmark feature of ASD. Our studies suggest that a valproic acid (VPA) rat model of ASD exhibits an enlargement of the amygdala as compared to controls rats, similar to that observed in adolescent ASD individuals. Since recent research suggests that altered neuronal development and morphology, as seen in ASD, may result from a common post-transcriptional process that is under tight regulation by microRNAs (miRs), we examined genome-wide transcriptomics expression in the amygdala of rats prenatally exposed to VPA, and detected elevated miR-181c and miR-30d expression levels as well as dysregulated expression of their cognate mRNA targets encoding proteins involved in neuronal system development. Furthermore, selective suppression of miR-181c function attenuates neurite outgrowth and branching, and results in reduced synaptic density in primary amygdalar neurons in vitro. Collectively, these results implicate the small non-coding miR-181c in neuronal morphology, and provide a framework of understanding how dysregulation of a neurodevelopmentally relevant miR in the amygdala may contribute to the pathophysiology of ASD.
Collapse
Affiliation(s)
- N F M Olde Loohuis
- Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - K Kole
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands
| | - J C Glennon
- Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - P Karel
- Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - G Van der Borg
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands
| | - Y Van Gemert
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands
| | - D Van den Bosch
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands
| | - J Meinhardt
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands
| | - A Kos
- Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - F Shahabipour
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands
| | - P Tiesinga
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands
| | - H van Bokhoven
- Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands; Department of Human Genetics, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - G J M Martens
- Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behavior, Nijmegen Centre for Molecular Life Sciences (NCMLS), Radboud University Nijmegen, Nijmegen, The Netherlands
| | - B B Kaplan
- Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA
| | - J R Homberg
- Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands
| | - A Aschrafi
- Department of Neuroinformatics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, Nijmegen, The Netherlands.
| |
Collapse
|
|
30
|
Bell JL, Turlapati R, Liu T, Schulte JH, Hüttelmaier S. IGF2BP1 harbors prognostic significance by gene gain and diverse expression in neuroblastoma. J Clin Oncol 2015; 33:1285-93. [PMID: 25753434 DOI: 10.1200/jco.2014.55.9880] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
PURPOSE Chromosomal 17q21-ter gain in neuroblastoma is both a common and prognostically significant event. The insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) gene is located near the proximal edge of this region. Here, its prognostic value is evaluated in neuroblastoma. METHODS The mRNA expression of IGF2BP family members was first evaluated by microarray data sets. In addition, in a separate cohort of 69 tumors, IGF2BP1 gene copy number, mRNA, and protein abundance were determined and compared with clinical parameters. RESULTS In two independent microarray data sets, 77% to 100% of tumors had substantial IGF2BP1 mRNA levels measured. High IGF2BP1 transcript abundance was significantly associated with stage 4 tumors (P < .001) and decreased patient survival (P < .001). IGF2BP1 was also associated with MYCN gene amplification and MYCN mRNA abundance. In the 69 neuroblastoma samples, IGF2BP1 DNA copy number (increased in 84% of tumors), mRNA, and protein abundance were significantly higher in stage 4 compared with stage 1 tumors. Importantly, IGF2BP1 protein levels were associated with lower overall patient survival (P = .012) and positively correlated with MYCN mRNA, even when excluding MYCN-amplified tumors. Moreover, IGF2BP1 clearly affected MYCN expression and neuroblastoma cell survival in vitro. CONCLUSION In neuroblastoma, IGF2BP1 was expressed in the majority of neuroblastoma specimens analyzed and was associated with lower overall patient survival and MYCN abundance. These data demonstrate that IGF2BP1 is a potential oncogene and an independent negative prognostic factor in neuroblastoma.
Collapse
Affiliation(s)
- Jessica L Bell
- Jessica L. Bell, Raseswari Turlapati, and Stefan Hüttelmaier, Martin Luther University Halle-Wittenberg, Halle; Johannes H. Schulte, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, and University Children's Hospital Essen, Essen; Johannes H. Schulte, German Cancer Consortium and German Cancer Research Center, Heidelberg, Germany; and Tao Liu, Children's Cancer Institute Australia for Medical Research and University of New South Wales, Randwick, New South Wales, Australia
| | - Raseswari Turlapati
- Jessica L. Bell, Raseswari Turlapati, and Stefan Hüttelmaier, Martin Luther University Halle-Wittenberg, Halle; Johannes H. Schulte, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, and University Children's Hospital Essen, Essen; Johannes H. Schulte, German Cancer Consortium and German Cancer Research Center, Heidelberg, Germany; and Tao Liu, Children's Cancer Institute Australia for Medical Research and University of New South Wales, Randwick, New South Wales, Australia
| | - Tao Liu
- Jessica L. Bell, Raseswari Turlapati, and Stefan Hüttelmaier, Martin Luther University Halle-Wittenberg, Halle; Johannes H. Schulte, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, and University Children's Hospital Essen, Essen; Johannes H. Schulte, German Cancer Consortium and German Cancer Research Center, Heidelberg, Germany; and Tao Liu, Children's Cancer Institute Australia for Medical Research and University of New South Wales, Randwick, New South Wales, Australia
| | - Johannes H Schulte
- Jessica L. Bell, Raseswari Turlapati, and Stefan Hüttelmaier, Martin Luther University Halle-Wittenberg, Halle; Johannes H. Schulte, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, and University Children's Hospital Essen, Essen; Johannes H. Schulte, German Cancer Consortium and German Cancer Research Center, Heidelberg, Germany; and Tao Liu, Children's Cancer Institute Australia for Medical Research and University of New South Wales, Randwick, New South Wales, Australia
| | - Stefan Hüttelmaier
- Jessica L. Bell, Raseswari Turlapati, and Stefan Hüttelmaier, Martin Luther University Halle-Wittenberg, Halle; Johannes H. Schulte, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, and University Children's Hospital Essen, Essen; Johannes H. Schulte, German Cancer Consortium and German Cancer Research Center, Heidelberg, Germany; and Tao Liu, Children's Cancer Institute Australia for Medical Research and University of New South Wales, Randwick, New South Wales, Australia.
| |
Collapse
|
|
31
|
Abstract
Neuroblastoma is the most common extracranial solid tumor of infancy. Amplification of MYCN oncogene is found in approximately 20 % of all neuroblastoma patients and correlates with advanced disease stages, rapid tumor progression, and poor prognosis, making this gene an obvious therapeutic target. However, being a transcriptional factor MYCN is difficult for pharmacological targeting, and there are currently no clinical trials aiming MYCN protein directly. Here we describe an alternative approach to address deregulated MYCN expression. In particular, we focus on the role of a 3′ untranslated region (3′UTR) of the MYCN gene in the modulation of its mRNA fate and identification of compounds able to affect it. The luciferase reporter construct with the full length MYCN 3′UTR was generated and subsequently integrated in the CHP134 neuroblastoma cell line. After validation, the assay was used to screen a 2000 compound library. Molecules affecting luciferase activity were checked for reproducibility and counter-screened for promoter effects and cytotoxic activity resulting in selection of four hits. We propose this cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms. Identification of such compounds could potentially result in development of clinically relevant therapeutics for various diseases including neuroblastoma.
Collapse
|
|
32
|
López-Doval S, Salgado R, Fernández-Pérez B, Lafuente A. Possible role of serotonin and neuropeptide Y on the disruption of the reproductive axis activity by perfluorooctane sulfonate. Toxicol Lett 2015; 233:138-47. [PMID: 25623392 DOI: 10.1016/j.toxlet.2015.01.012] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2014] [Revised: 01/20/2015] [Accepted: 01/21/2015] [Indexed: 01/09/2023]
Abstract
Perfluorooctane sulfonate (PFOS) is an endocrine disruptor, whose exposure can induce several alterations on the reproductive axis activity in males during adulthood. This study was undertaken to evaluate the possible role of serotonin and neuropeptide Y (NPY) on the disruption of the hypothalamic-pituitary-testicular (HPT) axis induced by PFOS in adult male rats. For that, adult male rats were orally treated with 0.5; 1.0; 3.0 and 6.0mg of PFOS/kg/day for 28 days. After PFOS exposure, serotonin concentration increased in the anterior and mediobasal hypothalamus as well as in the median eminence. The metabolism of this amine (expressed as the ratio 5-hydroxyindolacetic acid (5-HIAA)/serotonin) was diminished except in the anterior hypothalamus, with the doses of 3.0 and 6.0mg/kg/day, being this dose 0.5mg/kg/day in the median eminence. In general terms, PFOS-treated rats presented a decrease of the hypothalamic concentration of the gonadotropin releasing hormone (GnRH) and NPY. A diminution of the serum levels of the luteinizing hormone (LH), testosterone and estradiol were also shown. These results suggest that both serotonin and NPY could be involved in the inhibition induced by PFOS on the reproductive axis activity in adult male rats.
Collapse
Affiliation(s)
- S López-Doval
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain
| | - R Salgado
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain
| | - B Fernández-Pérez
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain
| | - A Lafuente
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain.
| |
Collapse
|
|
33
|
Mora J, Cruz O, Lavarino C, Rios J, Vancells M, Parareda A, Salvador H, Suñol M, Carrasco R, Guillen A, Mañé S, de Torres C. Results of induction chemotherapy in children older than 18 months with stage-4 neuroblastoma treated with an adaptive-to-response modified N7 protocol (mN7). Clin Transl Oncol 2015; 17:521-9. [PMID: 25596034 DOI: 10.1007/s12094-014-1273-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2014] [Accepted: 12/24/2014] [Indexed: 12/17/2022]
Abstract
PURPOSE We report the response rate in children older than 18 months with stage 4 Neuroblastoma, using a modified dose-intensive, response-adaptive, induction mN7 protocol. METHODS From 2005 to 2012, 24 patients were treated with the mN7 protocol. Phase 1 included five MSKCC N7 cycles and surgery and two high-dose cyclophosphamide-topotecan (HD-CT) cycles for those who did not achieve complete remission (CR) and negative bone marrow (BM) minimal residual disease (MRD) status (CR+MRD-). Phase 2 consisted of myeloablative doses of topotecan, thiotepa and carboplatin plus hyperfractionated RT. Phase 3 included isotretinoin and 3F8 immunotherapy plus GM-CSF. BM MRD was monitored using GD2 synthase, PHOX2B and cyclin D1 mRNAs. RESULTS After 3 cycles, all patients showed BM complete histological clearance and 6 (25 %) were MRD-. Twenty of 21 s-look surgeries achieved macroscopic complete resection. After 5 cycles and surgery, (123)I-MIBG scan was negative in 15 (62.5 %) cases, BM disease by histology was negative in 23 (96 %) and 10 (42 %) patients were MRD-. Twelve (50 %) pts were in CR, 2 in very good partial response (VGPR), 9 partial response (PR) and one had progressive disease. With 2 HD-CT extra cycles, 17 (71 %) pts achieved CR+MRD- status moving to phase 2. Overall and event-free survival at 3 years for the 17 patients who achieved CR+MRD- is 65 and 53 %, respectively, median follow-up 47 months. Seven (29 %) patients never achieved CR+MRD-. Univariate Cox regression analysis shows CR+MRD- status after mN7 induction as the only statistically significant prognostic factor to predict overall survival. CONCLUSIONS mN7 induction regimen produced a CR+MRD- rate of 71 %. CR+MRD- status following induction was the only predictive marker of long-term survival.
Collapse
Affiliation(s)
- J Mora
- Department of Oncology, Hospital Sant Joan de Déu, Barcelona, Passeig Sant Joan de Déu, 2, Esplugues de Llobregat, 08950, Barcelona, Spain,
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
34
|
Wang X, Jiang D, Yang D. Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR assay. Appl Biochem Biotechnol 2014; 175:996-1006. [PMID: 25351627 DOI: 10.1007/s12010-014-1322-3] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2014] [Accepted: 10/15/2014] [Indexed: 11/29/2022]
Abstract
The selection of homozygous lines is a crucial step in the characterization of newly generated transgenic plants. This is particularly time- and labor-consuming when transgenic stacking is required. Here, we report a fast and accurate method based on quantitative real-time PCR with a rice gene RBE4 as a reference gene for selection of homozygous lines when using multiple transgenic stacking in rice. Use of this method allowed can be used to determine the stacking of up to three transgenes within four generations. Selection accuracy reached 100 % for a single locus and 92.3 % for two loci. This method confers distinct advantages over current transgenic research methodologies, as it is more accurate, rapid, and reliable. Therefore, this protocol could be used to efficiently select homozygous plants and to expedite time- and labor-consuming processes normally required for multiple transgene stacking. This protocol was standardized for determination of multiple gene stacking in molecular breeding via marker-assisted selection.
Collapse
Affiliation(s)
- Xianghong Wang
- State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China
| | | | | |
Collapse
|
|
35
|
López-Doval S, Salgado R, Pereiro N, Moyano R, Lafuente A. Perfluorooctane sulfonate effects on the reproductive axis in adult male rats. ENVIRONMENTAL RESEARCH 2014; 134:158-168. [PMID: 25171141 DOI: 10.1016/j.envres.2014.07.006] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Revised: 07/08/2014] [Accepted: 07/12/2014] [Indexed: 06/03/2023]
Abstract
Perfluorooctane sulfonate (PFOS) is a neurotoxic agent and it can disrupt the endocrine system activity. This work was undertaken to evaluate the possible effects of PFOS exposure on the hypothalamic-pituitary-testicular axis (HPT) in adult male rats, and to evaluate the possible morphological alterations induced by PFOS in the endocrine tissues of this axis. Adult male rats were orally treated with 0.5; 1.0; 3.0 and 6.0 mg of PFOS/kg/day for 28 days. After PFOS exposure, hypothalamic noradrenaline concentration increased in the anterior hypothalamus and in the median eminence, not changing in the mediobasal hypothalamus. PFOS treated rats presented a decrease of the gonadotropin releasing hormone (GnRH) gene expression, increasing the mRNA levels of the luteinizing hormone (LH) in rats treated with all doses administered except with the dose of 6 mg/kg/day. PFOS also induced a raise of the follicle stimulating hormone (FSH) gene expression in the animals exposed to 0.5 and 1.0 mg of PFOS/kg/day. After PFOS exposure, hypothalamic GnRH concentration was modified, LH and testosterone release was inhibited and FSH secretion was stimulated. Moreover, PFOS induced several histopathological alterations in the hypothalamus, pituitary gland and testis. The results obtained in the present study suggest in general terms that PFOS can inhibit the physiological activity of the reproductive axis in adult male rats, which could be explained, at least in part, by the structural alterations showed in the animals exposed to this chemical: very dense chromatin, condensed ribosomes and a loss of the morphology in the hypothalamus; a degeneration of the gonadotrophic cells, as well as a loss and degeneration of the spermatozoids and a very marked edema in the testis.
Collapse
Affiliation(s)
- S López-Doval
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain
| | - R Salgado
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain
| | - N Pereiro
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain
| | - R Moyano
- Department of Pharmacology, Toxicology and Legal and Forensic Medicine, Veterinary Faculty, University of Córdoba, Córdoba 14071, Spain
| | - A Lafuente
- Laboratory of Toxicology, Sciences School, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain.
| |
Collapse
|
|
36
|
Dirse V, Burnyte B, Gineikiene E, Griskevicius L, Utkus A. A novel de novo 2.5 Mb microdeletion of 7q22.1 harbours candidate gene for neurobehavioural disorders and mental retardation. J Genet 2014; 93:501-3. [PMID: 25189247 DOI: 10.1007/s12041-014-0369-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Affiliation(s)
- Vaidas Dirse
- Hematology, Oncology and Transfusion Medicine Center, Vilnius University Hospital Santariskiu Klinikos, Vilnius LT-08661, Lithuania.
| | | | | | | | | |
Collapse
|
|
37
|
Wang X, Wang Y, Huang H, Chen B, Chen X, Hu J, Chang T, Lin RJ, Yee JK. Precise gene modification mediated by TALEN and single-stranded oligodeoxynucleotides in human cells. PLoS One 2014; 9:e93575. [PMID: 24691488 PMCID: PMC3972112 DOI: 10.1371/journal.pone.0093575] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2013] [Accepted: 03/05/2014] [Indexed: 11/18/2022] Open
Abstract
The development of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms, speeds up the process of drug screening, and raises the feasibility of using cell replacement therapy in clinics. However, the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs) spurred interest due to the ease of assembly, high efficiency and faithful gene targeting. In this study, we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall, our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application.
Collapse
Affiliation(s)
- Xiaoling Wang
- Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, United States of America
| | - Yingjia Wang
- Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, United States of America
- Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, China
| | - He Huang
- Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, China
| | - Buyuan Chen
- Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, United States of America
- Department of Hematology, Union Hospital of Fujian Medical University, Fuzhou, Fujian, China
| | - Xinji Chen
- Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, United States of America
- Department of Hematology, Union Hospital of Fujian Medical University, Fuzhou, Fujian, China
| | - Jianda Hu
- Department of Hematology, Union Hospital of Fujian Medical University, Fuzhou, Fujian, China
| | - Tammy Chang
- Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, United States of America
| | - Ren-Jang Lin
- Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, California, United States of America
| | - Jiing-Kuan Yee
- Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, United States of America
- * E-mail:
| |
Collapse
|
|
38
|
Pereiro N, Moyano R, Blanco A, Lafuente A. Regulation of corticosterone secretion is modified by PFOS exposure at different levels of the hypothalamic-pituitary-adrenal axis in adult male rats. Toxicol Lett 2014; 230:252-62. [PMID: 24440345 DOI: 10.1016/j.toxlet.2014.01.003] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2013] [Revised: 11/25/2013] [Accepted: 01/02/2014] [Indexed: 01/29/2023]
Abstract
Perfluorooctane sulfonate (PFOS) is a fluorinated compound and a Persistent Organic Pollutant which can disrupt the endocrine system. This work was undertaken to evaluate the possible effects of PFOS exposure on the regulation of corticosterone secretion in adrenal and pituitary glands and at hypothalamic level in adult male rat, and to evaluate the possible morphological alterations induced by PFOS in this endocrine tissue. Adult male rats were orally treated with 0.5, 1.0, 3.0 and 6.0 mg of PFOS/kg/day for 28 days. Corticosterone, adrenocorticotropic hormone (ACTH) and corticotrophin-releasing hormone (CRH) secretion decreased in PFOS-treated rats. After PFOS exposure, relative expression of adrenocorticotropic hormone receptor (ACTHr) and proopiomelanocortin (POMC) genes was increased in adrenal and in pituitary glands, respectively; while relative expression of ACTHr and CRH genes decreased in hypothalamus with the doses of 0.5 and 1.0 mg/kg/day. PFOS treatment increased relative nitric oxide synthase 1 and 2 (NOS1 and NOS2) gene expression in the adrenal gland, and incremented superoxide dismutase activity. PFOS exposure induces a global inhibition of the hypothalamic-pituitary-adrenal (HPA) axis activity, and small morphological changes were observed in adrenal zona fasciculata cells.
Collapse
Affiliation(s)
- N Pereiro
- Laboratory of Toxicology, Faculty of Sciences, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain
| | - R Moyano
- Department of Pharmacology, Toxicology and Legal and Forensic Medicine, Veterinary Faculty, University of Córdoba, 14071, Córdoba, Spain
| | - A Blanco
- Department of Comparative Pathology, Faculty of Veterinary Medicine, University of Córdoba, 14071 Córdoba, Spain
| | - A Lafuente
- Laboratory of Toxicology, Faculty of Sciences, University of Vigo, Las Lagunas S/n, 32004 Ourense, Spain.
| |
Collapse
|
|
39
|
Gupta H, Sakharwade SC, Angural A, Kotambail A, Bhat GK, Hande MH, D'Souza SC, Rao P, Kumari V, Saadi AV, Satyamoorthy K. Evidence for genetic linkage between a polymorphism in the GNAS gene and malaria in South Indian population. Acta Trop 2013; 128:571-7. [PMID: 23962387 DOI: 10.1016/j.actatropica.2013.08.005] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2013] [Revised: 07/08/2013] [Accepted: 08/12/2013] [Indexed: 12/12/2022]
Abstract
The complex imprinted GNAS locus which encodes G-alpha subunit (Gαs) is involved in a number of G-protein coupled signaling pathways in eukaryotic cells. Erythrocyte invasion by Plasmodium falciparum parasites is significantly regulated by protein of GNAS gene. This study was designed to evaluate the association between single nucleotide polymorphisms (SNPs) present in GNAS locus and susceptibility to malaria. In this case control study, individuals affected by P. falciparum malaria (n=230), Plasmodium vivax malaria (n=230) and normal controls (n=230) were tested for the association of eighteen (18) known SNPs to evaluate their role in the onset of the disease. There was no significant difference in genotype frequencies of all the SNPs tested between P. falciparum and P. vivax affected individuals. However, when Bonferroni correction for multiple comparisons were performed as a control, our results demonstrated alleles and genotypes of rs7121: C>T (NC_000020.10:g.57478807C>T), a silent polymorphism situated in the exon 5, were significantly (p<0.05) associated with susceptibility to malaria in the South Indians participants. Our results demonstrate that population specific polymorphisms that exist in GNAS gene may alter the risk of occurrence of malaria.
Collapse
|
|
40
|
A recombinant trans-membrane protein hMnSOD–R9 inhibits the proliferation of cervical cancer cells in vitro. Mol Cell Biochem 2013; 385:79-86. [DOI: 10.1007/s11010-013-1816-2] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Accepted: 09/13/2013] [Indexed: 12/30/2022]
|
|
41
|
Wang Y, Pijut PM. Isolation and characterization of a TERMINAL FLOWER 1 homolog from Prunus serotina Ehrh. TREE PHYSIOLOGY 2013; 33:855-65. [PMID: 23956129 DOI: 10.1093/treephys/tpt051] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/11/2023]
Abstract
Flowering control is one of the several strategies for gene containment of transgenic plants. TERMINAL FLOWER 1 (TFL1) is known to be involved in the transcriptional repression of genes for inflorescence development. Two TFL1 transcripts with different 3' UTR were cloned from black cherry (Prunus serotina Ehrh.) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Corresponding to the two TFL1 transcripts, two PsTFL1 gene sequences, 1248 bp and 1579 bp, were obtained and both contained the same 519 bp coding region which encoded a putative protein of 172 amino acid residues. The phylogenetic analysis of the amino acid sequences showed high identity of PsTFL1 to TFL1 orthologs of other Prunus species, including Yoshino cherry (Prunus × yedoensis Matsum.), peach (Prunus persica (L.) Batsch), apricot (Prunus armeniaca L.) and Japanese apricot (Prunus mume Sieb. et Zucc.). The real-time quantitative PCR detected a single copy of PsTFL1 gene sequences in the black cherry genome with two alleles. The gene expression of PsTFL1 was examined in several tissues including the stems, leaves, shoot tips, and vegetative and floral buds. The highest mRNA level was detected in shoot tips, and the lowest level in the leaves. Transgenic Arabidopsis thaliana (L.) Heynh. plants overexpressing PsTFL1 showed significantly delayed flowering. These plants also showed largely increased vegetative growth, plant height, number of nodes, trichome density, and the conversion of flower to shoot was observed at each node and shoot apex.
Collapse
Affiliation(s)
- Ying Wang
- Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center (HTIRC), Purdue University, 715 West State St., West Lafayette, IN 47907, USA
| | | |
Collapse
|
|
42
|
Van Mater D, Knelson EH, Kaiser-Rogers KA, Armstrong MB. Neuroblastoma in a pediatric patient with a microduplication of 2p involving theMYCNlocus. Am J Med Genet A 2013; 161A:605-10. [DOI: 10.1002/ajmg.a.35766] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2011] [Accepted: 10/15/2012] [Indexed: 11/11/2022]
|
|
43
|
Kempinski CF, Crowell SV, Smeeth C, Barth C. The novel Arabidopsis thaliana svt2 suppressor of the ascorbic acid-deficient mutant vtc1-1 exhibits phenotypic and genotypic instability. F1000Res 2013; 2:6. [PMID: 24627766 PMCID: PMC3938180 DOI: 10.12688/f1000research.2-6.v1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 12/19/2012] [Indexed: 12/13/2022] Open
Abstract
Ascorbic acid is a potent antioxidant that detoxifies reactive oxygen species when plants are exposed to unfavorable environmental conditions. In addition to its antioxidant properties, ascorbic acid and its biosynthetic precursors fulfill a variety of other physiological and molecular functions. A mutation in the ascorbic acid biosynthesis gene
VTC1, which encodes GDP-mannose pyrophosphorylase, results in conditional root growth inhibition in the presence of ammonium. To isolate suppressors of
vtc1-1, which is in the
Arabidopsis Columbia-0 background, seeds of the mutant were subjected to ethyl methanesulfonate mutagenesis. A suppressor mutant of
vtc1-1 2,
svt2, with wild-type levels of ascorbic acid and root growth similar to the wild type in the presence of ammonium was isolated. Interestingly,
svt2 has
Arabidopsis Landsberg
erecta features, although
svt2 is delayed in flowering and has an enlarged morphology. Moreover, the
svt2 genotype shares similarities with L
er polymorphism markers and sequences, despite the fact that the mutant derived from mutagenesis of Col-0
vtc1-1 seed. We provide evidence that
svt2 is not an artifact of the experiment, a contamination of L
er seed, or a result of outcrossing of the
svt2 mutant with L
er pollen. Instead, our results show that
svt2 exhibits transgenerational genotypic and phenotypic instability, which is manifested in a fraction of
svt2 progeny, producing revertants that have Col-like phenotypic and genotypic characteristics. Some of those Col-like revertants then revert back to
svt2-like plants in the subsequent generation. Our findings have important implications for undiscovered phenomena in transmitting genetic information in addition to the Mendelian laws of inheritance. Our results suggest that stress can trigger a genome restoration mechanism that could be advantageous for plants to survive environmental changes for which the ancestral genes were better adapted.
Collapse
Affiliation(s)
- Chase F Kempinski
- Department of Biology, West Virginia University, Morgantown, 26506, USA ; Department of Plant and Soil Sciences, University of Kentucky, Lexington, 40546, USA
| | - Samuel V Crowell
- Department of Biology, West Virginia University, Morgantown, 26506, USA ; Department of Plant Biology, Cornell University, Ithaca, 14853, USA
| | - Caleb Smeeth
- Department of Biology, West Virginia University, Morgantown, 26506, USA ; ACTION-Housing Inc., Pittsburgh, 15219, USA
| | - Carina Barth
- Department of Biology, West Virginia University, Morgantown, 26506, USA ; ConRuhr North America, New York, 10017, USA
| |
Collapse
|
|
44
|
Abstract
Members of the DEAD box family of RNA helicases are known to be involved in most cellular processes that require manipulation of RNA structure and, in many cases, exhibit other functions in addition to their established ATP-dependent RNA helicase activities. They thus play critical roles in cellular metabolism and in many cases have been implicated in cellular proliferation and/or neoplastic transformation. These proteins generally act as components of multi-protein complexes; therefore their precise role is likely to be influenced by their interacting partners and to be highly context-dependent. This may also provide an explanation for the sometimes conflicting reports suggesting that DEAD box proteins have both pro- and anti-proliferative roles in cancer.
Collapse
Affiliation(s)
- Frances V Fuller-Pace
- Division of Cancer Research, University of Dundee, Ninewells Hospital and Medical School, Dundee, Scotland.
| |
Collapse
|
|
45
|
Casalà C, Gil-Guiñón E, Ordóñez JL, Miguel-Queralt S, Rodríguez E, Galván P, Lavarino C, Munell F, de Alava E, Mora J, de Torres C. The calcium-sensing receptor is silenced by genetic and epigenetic mechanisms in unfavorable neuroblastomas and its reactivation induces ERK1/2-dependent apoptosis. Carcinogenesis 2012; 34:268-76. [PMID: 23108190 DOI: 10.1093/carcin/bgs338] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5'aza-2-deoxycitidine and/or trichostatin A restored CaSR expression in MYCN-amplified cell lines. Following 5'aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival.
Collapse
Affiliation(s)
- Carla Casalà
- Developmental Tumor Biology Laboratory, Hospital Sant Joan de Déu and Fundació Sant Joan de Déu, Barcelona, Spain
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
46
|
Ehlén Å, Rosselló CA, von Stedingk K, Höög G, Nilsson E, Pettersson HM, Jirström K, Alvarado-Kristensson M. Tumors with nonfunctional retinoblastoma protein are killed by reduced γ-tubulin levels. J Biol Chem 2012; 287:17241-17247. [PMID: 22493456 PMCID: PMC3366773 DOI: 10.1074/jbc.m112.357038] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
In various tumors inactivation of growth control is achieved by interfering with the RB1 signaling pathway. Here, we describe that RB1 and γ-tubulin proteins moderate each other's expression by binding to their respective gene promoters. Simultaneous reduction of RB1 and γ-tubulin protein levels results in an E2F1-dependent up-regulation of apoptotic genes such as caspase 3. We report that in various tumors types, there is an inverse correlation between the expression levels of γ-tubulin and RB1 and that in tumor cell lines with a nonfunctioning RB1, reduction of γ-tubulin protein levels leads to induction of apoptosis. Thus, the RB1/γ-tubulin signal network can be considered as a new target for cancer treatment.
Collapse
Affiliation(s)
- Åsa Ehlén
- Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Catalina A Rosselló
- Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Kristoffer von Stedingk
- Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Greta Höög
- Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Elise Nilsson
- Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Helen M Pettersson
- Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden
| | - Karin Jirström
- Department of Clinical Sciences, Pathology, Lund University, Skåne University Hospital, SE-221 85 Lund, Sweden
| | - Maria Alvarado-Kristensson
- Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, SE-205 02 Malmö, Sweden.
| |
Collapse
|
|
47
|
Ghassemifar S, Mendrysa SM. MDM2 antagonism by nutlin-3 induces death in human medulloblastoma cells. Neurosci Lett 2012; 513:106-10. [PMID: 22343310 DOI: 10.1016/j.neulet.2012.02.022] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2012] [Revised: 02/07/2012] [Accepted: 02/08/2012] [Indexed: 11/28/2022]
Abstract
A critical component of the cellular stress response, the p53 tumor suppressor protein must be functional for many cancer therapies to be effective. Adjuvant therapies that augment p53 function are predicted to sensitize tumor cells to cancer therapies that rely upon p53 for their efficacy. Of those strategies currently being explored to enhance p53 function, inhibition of the ubiquitin ligase, MDM2, a negative regulator of p53, has shown promise. Here, we investigated whether MDM2 antagonism might be effective in inducing cell death in human medulloblastoma (MB) cells. Nutlin-3, a small-molecule inhibitor of MDM2, potently induced apoptosis in MB cells with wild-type TP53. Moreover, nutlin-3 potentiated p53 activation and growth impairment of MB cells in combination with the classic DNA-damaging agent doxorubicin. Together, these results support the concept that MDM2 antagonists may be therapeutically beneficial for patients with MB tumors.
Collapse
Affiliation(s)
- Sara Ghassemifar
- Department of Basic Medical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA
| | | |
Collapse
|
|
48
|
Gadaleta A, Giancaspro A, Cardone MF, Blanco A. Real-time PCR for the detection of precise transgene copy number in durum wheat. Cell Mol Biol Lett 2011; 16:652-68. [PMID: 21922222 PMCID: PMC6275630 DOI: 10.2478/s11658-011-0029-5] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2011] [Accepted: 09/09/2011] [Indexed: 01/09/2023] Open
Abstract
Recent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of "estimating" copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.
Collapse
Affiliation(s)
- Agata Gadaleta
- Department of Environmental and Agro-Forestry Biology and Chemistry, Section of Genetics and Plant Breeding, University of Bari Aldo Moro, Via Amendola 165/A-70126, Bari, Italy.
| | | | | | | |
Collapse
|
|
49
|
Bae JY, Lee JS, Shin MH, Lee SH, Hwang IG. Effect of wash treatments on reducing human norovirus on iceberg lettuce and perilla leaf. J Food Prot 2011; 74:1908-11. [PMID: 22054192 DOI: 10.4315/0362-028x.jfp-11-063] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Human noroviruses (NoVs) are major causes of nonbacterial gastroenteritis; they are transmitted by food and water, as well as person-to-person. The consumption of contaminated raw or uncooked food such as vegetables and fruits has been identified as a common source of human NoV outbreaks. In an effort to understand the survival and persistence of human NoVs on fresh produce, the efficacy of washing treatments in the removal of human NoVs from vegetables was evaluated. This study used artificially contaminated vegetables (iceberg lettuce and perilla leaf), and washing was done with tap water for convenience. Wash treatments included immersion in water, rinsing with running water, and a combination of immersion and rinsing (treatments I to III, respectively). The effect of a class I detergent, a commercial product used for washing fruits and vegetables, was also evaluated (treatment IV). After the wash treatments, the remnants of human NoVs on samples were measured via real-time reverse transcriptase PCR. The results varied among treatments and by vegetable. For iceberg lettuce, a reduction of 0.9 log was noted in the treatment III group. The wash treatment was more effective in the perilla leaf samples: each treatment significantly reduced the numbers of human NoVs (0.69- to 1.29-log reduction). These data demonstrated that wash treatments reduced numbers of virus from the surfaces of the vegetables. Therefore, washing would seem to be a basic step in reducing numbers of virus in food preparation and in viral transmission routes.
Collapse
Affiliation(s)
- Ju-Yun Bae
- Food Microbiology Division, Food Safety Evaluation Department, National Institute of Food and Drug Safety Evaluation, 643 Yeonjae-ri, Gangoe-myon, Cheongwon-gun, Chungcheongbuk-do 363-951, Republic of Korea
| | | | | | | | | |
Collapse
|
|
50
|
Louis-Brennetot C, Coindre JM, Ferreira C, Pérot G, Terrier P, Aurias A. The CDKN2A/CDKN2B/CDK4/CCND1 pathway is pivotal in well-differentiated and dedifferentiated liposarcoma oncogenesis: an analysis of 104 tumors. Genes Chromosomes Cancer 2011; 50:896-907. [PMID: 21910158 DOI: 10.1002/gcc.20909] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2011] [Accepted: 06/26/2011] [Indexed: 12/30/2022] Open
Abstract
The MDM2 and CDK4 genes are the main targets of chromosome 12 amplification in well-differentiated and dedifferentiated liposarcomas. Nevertheless, around 10% of these tumors do not amplify CDK4. To find substitutive alterations of CDK4 amplification, we analyzed a large series of liposarcomas by array-CGH, real-time genomic PCR, gene expression array, and real-time RT-PCR. We demonstrate that an alteration in the CDKN2A/CDKN2B/CDK4/CCND1 pathway is present in almost all cases without CDK4 amplification, thereby confirming the pivotal role of this pathway in liposarcoma oncogenesis. Moreover, we show that cell cycle and differentiation are driven by a subtle and complex balance between members of this pathway. Finally, we demonstrate that in tumors without amplification/overexpression of CDK4, the chromosome 1q21-1q23 region is a preferential partner of chromosome 12 amplicon, suggesting that the mechanism of amplification is slightly different in this group of tumors.
Collapse
|