Expression of NAD(P)H:Quinone oxidoreductase 1 in HeLa cells -: Role of hydrogen peroxide and growth phase

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 muM) produced a significant increase of NQO1 (up to 16-fold at 12 muM) 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.