JBC Invitrogen Ultrasensitive Cytokine Assays

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J Biol Chem, Vol. 274, Issue 53, 37679-37684, December 31, 1999

Endogenous Endothelial Cell Nitric-oxide Synthase Modulates Apoptosis in Cultured Breast Cancer Cells and Is Transcriptionally Regulated by p53*

Kirsten MortensenDagger , Jan Skouv§, David M. Hougaard, and Lars-Inge LarssonDagger ∥

From the Dagger  Division of Cell Biology, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg, DK-2950 Denmark, § Exiqon A/S, Vedbæk, Denmark, and the  Department of Clinical Biochemistry, Statens Serum Institut, DK-2300 Copenhagen, Denmark

Nitric oxide can both stimulate and suppress apoptosis. By reverse transcriptase-polymerase chain reaction and sequencing we show that human breast cancer (MCF-7) cells express endothelial cell nitric-oxide synthase (ecNOS), but not other nitric-oxide synthase isoforms. Inhibition of ecNOS activity in MCF-7 cells increased tumor cell apoptosis, and this effect was also seen following treatment with an NO scavenger. In addition, low concentrations of the NO donor sodium nitroprusside inhibited, whereas high concentrations stimulated MCF-7 cell apoptosis. The ecNOS promoter was found to contain a specific binding site for the apoptosis-regulating protein p53. In co-transfection studies wild-type, but not mutant, p53 down-regulated transcription of an ecNOS promoter-luciferase reporter gene construct. In addition, NO donors up-regulated p53 protein levels in MCF-7 cells. These data point to a previously unrecognized p53-dependent regulation of ecNOS expression that may be important both for regulating apoptosis and for avoiding the generation of genotoxic quantities of NO.


* This work was supported by the Danish Medical Research Council, the Danish Cancer Society, and the Danish National Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Div. of Cell Biology, Inst. of Anatomy and Physiology, Royal Veterinary and Agricultural University, Gronnegaardsvej 7, DK-1870 Frederiksberg C, Denmark. Tel.: 45-35-28-28-51; Fax: 45-35-28-25-47; E-mail: Lail@kvl.dk.


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.



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