David Barzan1,2, Patrick Maier3, W Jens Zeller4, Frederik Wenz3, Carsten Herskind5,6. 1. Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany. david.barzan@medma.uni-heidelberg.de. 2. Klinik für Strahlentherapie und Radioonkologie, UMM, Universitätsmedizin Mannheim, Theodor-Kutzer-Ufer 1-3, 68153, Mannheim, Germany. david.barzan@medma.uni-heidelberg.de. 3. Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany. 4. Pharmacology of Cancer Treatment, German Cancer Research Center, Heidelberg, Germany. 5. Department of Radiation Oncology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany. carsten.herskind@medma.uni-heidelberg.de. 6. Klinik für Strahlentherapie und Radioonkologie, UMM, Universitätsmedizin Mannheim, Theodor-Kutzer-Ufer 1-3, 68153, Mannheim, Germany. carsten.herskind@medma.uni-heidelberg.de.
Abstract
BACKGROUND AND PURPOSE: The transmembrane protein caveolin-1 (CAV1) is an essential component of caveolae, small membrane invaginations involved in vesicle formation. CAV1 plays a role in signal transduction, tumor suppression and oncogene transformation. Previous studies with CAV1 knockout mice and CAV1 knockdown in pancreatic tumor cells implicated CAV1 in mediating radioresistance. The aim of this work was to test the effect of CAV1 overexpression after irradiation in human cells lacking endogenous CAV1 expression. MATERIAL AND METHODS: Human CAV1 was overexpressed in lymphoblastoid TK6 cells (TK6-wt) using a eukaryotic expression plasmid, pCI-CAV1, or a lentiviral SIN (self-inactivating) vector, HR'SIN-CAV1. CAV1 expression was verified in TK6 cells with Western blot analysis or intracellular FACS (fluorescence-activated cell sorting) staining. The effect of CAV1 on proliferation kinetics after irradiation of TK6 cells was measured with a growth assay. RESULTS: TK6-wt showed no detectable endogenous CAV1 expression. Lentivirally mediated transduction with HR'SIN-CAV1 (TK6-CAV1) resulted in a considerably stronger CAV1 expression in comparison to TK6 cells electroporated with pCI-CAV1. Intracellular FACS analysis showed that 90% of transduced cells expressed CAV1. CAV1 enhanced early proliferation of TK6 cells after irradiation with a dose of 2 Gy, whereas proliferation of unirradiated cells was not affected. CAV1 also protected cells after irradiation with 4 Gy. This radioprotective effect was supported by a reduction of radiation-induced apoptosis. CONCLUSION: A model system for expression of exogenous CAV1 by stable lentiviral transduction of TK6 cells was established. Functional assays demonstrated enhanced early proliferation by CAV1 expression in TK6 cells after irradiation with clinically relevant doses supporting the role of CAV1 as a prosurvival factor.
BACKGROUND AND PURPOSE: The transmembrane protein caveolin-1 (CAV1) is an essential component of caveolae, small membrane invaginations involved in vesicle formation. CAV1 plays a role in signal transduction, tumor suppression and oncogene transformation. Previous studies with CAV1 knockout mice and CAV1 knockdown in pancreatic tumor cells implicated CAV1 in mediating radioresistance. The aim of this work was to test the effect of CAV1 overexpression after irradiation in human cells lacking endogenous CAV1 expression. MATERIAL AND METHODS:HumanCAV1 was overexpressed in lymphoblastoid TK6 cells (TK6-wt) using a eukaryotic expression plasmid, pCI-CAV1, or a lentiviral SIN (self-inactivating) vector, HR'SIN-CAV1. CAV1 expression was verified in TK6 cells with Western blot analysis or intracellular FACS (fluorescence-activated cell sorting) staining. The effect of CAV1 on proliferation kinetics after irradiation of TK6 cells was measured with a growth assay. RESULTS: TK6-wt showed no detectable endogenous CAV1 expression. Lentivirally mediated transduction with HR'SIN-CAV1 (TK6-CAV1) resulted in a considerably stronger CAV1 expression in comparison to TK6 cells electroporated with pCI-CAV1. Intracellular FACS analysis showed that 90% of transduced cells expressed CAV1. CAV1 enhanced early proliferation of TK6 cells after irradiation with a dose of 2 Gy, whereas proliferation of unirradiated cells was not affected. CAV1 also protected cells after irradiation with 4 Gy. This radioprotective effect was supported by a reduction of radiation-induced apoptosis. CONCLUSION: A model system for expression of exogenous CAV1 by stable lentiviral transduction of TK6 cells was established. Functional assays demonstrated enhanced early proliferation by CAV1 expression in TK6 cells after irradiation with clinically relevant doses supporting the role of CAV1 as a prosurvival factor.
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