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J. Biol. Chem., Vol. 276, Issue 48, 44379-44384, November 30, 2001

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Expression of NAD(P)H:Quinone Oxidoreductase 1 in HeLa Cells
ROLE OF HYDROGEN PEROXIDE AND GROWTH PHASE^*

Rosario I. Bello, Consuelo Gómez-Díaz^§, Francisco Navarro^¶, Francisco J. Alcaín, and José M. Villalba

From the Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Universidad de Córdoba, Cordoba, 14071 Spain

The aim of this work was to study the role of H₂O₂ in the regulation of NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC 1.6.99.2) with relation to cell density of HeLa cells cultures and the function played by NQO1 in these cells. Levels of NQO1 activity were much higher (40-fold) in confluent HeLa cells than in sparse cells, the former cells being much more resistant to H₂O₂. Addition of sublethal concentrations of H₂O₂ (up to 24 µM) produced a significant increase of NQO1 (up to 16-fold at 12 µM) in sparse cells but had no effect in confluent cells. When cells reached confluency in the presence of pyruvate, a H₂O₂ scavenger, NQO1 activity was decreased compared with cultures grown to confluency without pyruvate. Inhibition of quinone reductases by dicumarol substantially decreased viability of confluent cells in serum-free medium. This is the first demonstration that regulation of NQO1 expression by H₂O₂ is dependent on the cell density in HeLa cells and that endogenous generation of H₂O₂ participates in the increase of NQO1 activity as cell density is higher. This enzyme is required to promote survival of confluent cells.

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