Carcinogenesis Advance Access published online on June 15, 2006
Carcinogenesis, doi:10.1093/carcin/bgl111
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1 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112; Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112
* To whom correspondence should be addressed. Previous studies demonstrate that the covalent modification of thioredoxin reductase (TrxR) by both endogenous and exogenous electrophiles results in disruption of the conformation of the tumor suppressor protein p53. Here we report that the loss of normal cellular TrxR enzymatic activity by electrophilic modification or deletion of the C-terminal catalytic selenocysteine residue has functional consequences that are distinct from those resulting from depletion of TrxR protein in human RKO colon cancer cells. A thorough kinetic analysis was performed on purified TrxR in order to characterize the mechanism of its inhibition by electrophiles. Furthermore, electrospray mass spectrometry confirmed the alkylation of TrxR by lipid electrophiles and liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS) identified the C-terminus as one target for alkylation. Then the consequences of TrxR modification by electrophiles on p53 conformation, transactivation, and on apoptosis were compared and contrasted to the effects of depletion of TrxR protein by treatment of cells with small interfering RNA directed against TrxR1. We found that cells depleted of TrxR were actually less sensitive to electrophile-induced disruption of p53 conformation and apoptosis than were cells expressing normal levels of TrxR. When RKO cells depleted of wild-type TrxR were transfected with C-terminal mutants of TrxR lacking the catalytic selenocysteine, p53 was found to be conformationally deranged, similar to cells treated with electrophiles. These results lead us to conclude that C-terminal modification of TrxR is both necessary and sufficient for the disruption of p53 and for the induction of apoptosis. Endogenous lipid electrophiles have been our primary focus; however, metabolic activation of hormones can generate endogenous mutagens, and we demonstrate that estrone-quinone attenuates p53 function in human MCF-7 cells.
Received November 3, 2005
Revised May 26, 2006
Accepted May 29, 2006
CARCINOGENESIS
Thioredoxin reductase is required for the inactivation of tumor suppressor p53 and for apoptosis induced by endogenous electrophiles
Pamela B. Cassidy 1,
Kornelia Edes 2,
Chad C. Nelson 3,
Krishna Parsawar 4,
F. A. Fitzpatrick 1,
and
Philip J. Moos 5 *
2 Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112
3 Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112; Mass Spectrometry and Proteomics Core Facility, University of Utah, Salt Lake City, UT 84112
4 Mass Spectrometry and Proteomics Core Facility, University of Utah, Salt Lake City, UT 84112
5 Department of Pharmacology & Toxicology, University of Utah, Salt Lake City, UT 84112
Philip J. Moos, E-mail: philip.moos{at}pharm.utah.edu
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