Enhancement of gene expression under hypoxic conditions using fragments of the human vascular endothelial growth factor and the erythropoietin genes |
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| Institution: | 1. Department of Radiology, Sakyo-ku, Kyoto, Japan;2. Department of Molecular Oncology, Faculty of Medicine, Kyoto University; 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto, Japan;1. Infectious Diseases Department and “Lluita contra la SIDA” Foundation, University Hospital Germans Trias i Pujol, Badalona, Spain;2. Universitat Autònoma de Barcelona, Spain;3. Fundació Clinic per a la Recerca Biomèdica, Hospital Clinic/IDIBAPS, Barcelona, Spain;4. Infectious Diseases, St. Louis Hospital, Paris Diderot University, Paris, France;5. Infectious Diseases, Hospital La Paz, Madrid, Spain;6. HIV Unit, Infectious Disease Service, IDIBELL-Hospital Universitari de Bellvitge, L’Hospitalet, Barcelona, Spain;7. UAB, UVIC-UCC, IEC, Spain;8. Hospital de Sant Pau, Barcelona, Spain;9. Infectious Diseases & AIDS Units, Hospital Clinic/IDIBAPS, University of Barcelona, Barcelona, Spain;1. Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, South Korea;1. Department of Diagnostic Radiology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 123 Ta-Pei Road, Niao-Sung, Kaohsiung 83305 Taiwan;2. Department of Surgery, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 123 Ta-Pei Road, Niao-Sung, Kaohsiung 83305 Taiwan;1. Department of Pathology and Laboratory Medicine, University of California Davis, Sacramento, CA, USA;2. Department of Public Health Sciences, Division of Biostatistics, University of California Davis, Davis, CA, USA;1. Thoraxcenter, Erasmus MC, Rotterdam, The Netherlands;2. Duke Clinical Research Institute, Durham, North Carolina;3. Flinders University and Medical Centre, Bedford Park, Adelaide, Australia;4. Division of Cardiology, University of Alberta, Edmonton, Canada;5. Department of Cardiology, University of Leuven, Leuven, Belgium;6. Fondazione IRCCS Policlinico San Matteo, Pavia, Italy;7. Green Lane Cardiovascular Service, Auckland, New Zealand;8. University Hospital Kralovske Vinohrady, Charles University, Prague, Czech Republic;9. Department of Medicine, Stanford University, Stanford, California;10. Hospital Universitari de Bellvitge, Universitat de Barcelona, Barcelona, Spain;11. Bayer HealthCare Pharmaceuticals Inc., Whippany, New Jersey;12. Department of Medical Sciences, Uppsala Clinical Research Center, Uppsala, Sweden;13. Merck & Co., Whitehouse Station, New Jersey;14. Division of Cardiovascular Medicine, Gill Heart Institute, University of Kentucky, Lexington, Kentucky |
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| Abstract: | Purpose: Selective gene expression in response to tumor hypoxia may provide new avenues, not only for radiotherapy and chemotherapy, but also for gene therapy. In this study, we have assessed the extent of hypoxia responsiveness of various DNA constructs by the luciferase assay to help design vectors suitable for cancer therapy.Materials and Methods: Reporter plasmids were constructed with fragments of the human vascular endothelial growth factor (VEGF) and the erythropoietin (Epo) genes encompassing the putative hypoxia-responsive elements (HRE) and the pGL3 promoter vector. Test plasmids and the control pRL-CMV plasmid were cotransfected into tumor cells by the calcium phosphate method. After 6 h hypoxic treatment, the reporter assay was performed.Results: The construct pGL3/VEGF containing the 385 bp fragment of the 5’ flanking region in human VEGF gene showed significant increases in luciferase activity in response to hypoxia. The hypoxic/aerobic ratios were about 3–4, and 8–12 for murine and human tumor cells, respectively. Despite the very high degree of conservation among the HREs of mammalian VEGF genes, murine cells showed lower responsiveness than human cells. We next tested the construct pGL3/Epo containing the 150 bp fragment of the 3’ flanking region in the Epo gene. Luciferase activity of pGL3/Epo was increased with hypoxia only in human cell lines. The insertion of 5 copies of the 35-bp fragments derived from the VEGF HREs and 32 bp of the E1b minimal promoter resulted in maximal enhancement of hypoxia responsiveness.Conclusions: The constructs with VEGF or Epo fragments containing HRE may be useful for inducing specific gene expression in hypoxic cells. Especially, the application of multiple copies of the HREs and an E1b minimal promoter appears to have the advantage of great improvement in hypoxia responsiveness. |
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