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J. Biol. Chem., Vol. 279, Issue 52, 54510-54517, December 24, 2004

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Overexpression of Enzymatically Active Human Cytosolic and Mitochondrial Thioredoxin Reductase in HEK-293 Cells

EFFECT ON CELL GROWTH AND DIFFERENTIATION^*

Ivan Nalvarte, Anastasios E. Damdimopoulos, Christina Nystöm, Tomas Nordman¶, Antonio Miranda-Vizuete, Jerker M. Olsson, Lennart Eriksson, Mikael Björnstedt, Elias S. J. Arnér¶, and Giannis Spyrou||**

From the Department of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, SE-141 57 Huddinge, Sweden, the Department of Laboratory Medicine, Division of Pathology, F46, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-141 86 Huddinge, Sweden, the ^¶Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden, and ^||Institute of Biomedical Research, Academy of Athens, Soranou Efessiou 4, 11527 Athens, Greece

The mammalian thioredoxin reductases (TrxR) are selenoproteins containing a catalytically active selenocysteine residue (Sec) and are important enzymes in cellular redox control. The cotranslational incorporation of Sec, necessary for activity, is governed by a stem-loop structure in the 3'-untranslated region of the mRNA and demands adequate selenium availability. The complicated translation machinery required for Sec incorporation is a major obstacle in isolating mammalian cell lines stably overexpressing selenoproteins. In this work we report on the development and characterization of stably transfected human embryonic kidney 293 cells that overexpress enzymatically active selenocysteine-containing cytosolic TrxR1 or mitochondrial TrxR2. We demonstrate that the overexpression of selenium-containing TrxR1 results in lower expression and activity of the endogenous selenoprotein glutathione peroxidase and that the activity of overexpressed TrxRs, rather than the protein amount, can be increased by selenium supplementation in the cell growth media. We also found that the TrxR-overexpressing cells grew slower over a wide range of selenium concentrations, which was an effect apparently not related to increased apoptosis nor to fatally altered intracellular levels of reactive oxygen species. Most surprisingly, the TrxR1- or TrxR2-overexpressing cells also induced novel expression of the epithelial markers CK18, CK-Cam5.2, and BerEP4, suggestive of a stimulation of cellular differentiation.

Received for publication, July 27, 2004 , and in revised form, October 6, 2004.

^* This work was supported by Åke Wibergsstiftelse, Karolinska Institutet, Södertörns Högskola, and the Swedish Medical Research Council Projects 13X-10370, 03P-14096-01A, and 03X-14041-01A. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Biosciences at Novum, Center for Biotechnology, Karolinska Institutet, SE-141 57 Huddinge, Sweden. Tel.: 46-8-6089162; Fax: 46-8-7745538; E-mail: giannis.spyrou{at}cbt.ki.se.

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