Abstract
The aim of this work was to study the role of H(2)O(2) in the regulation of NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase, EC ) 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 H(2)O(2). Addition of sublethal concentrations of H(2)O(2) (up to 24 microm) produced a significant increase of NQO1 (up to 16-fold at 12 microm) in sparse cells but had no effect in confluent cells. When cells reached confluency in the presence of pyruvate, a H(2)O(2) 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 H(2)O(2) is dependent on the cell density in HeLa cells and that endogenous generation of H(2)O(2) participates in the increase of NQO1 activity as cell density is higher. This enzyme is required to promote survival of confluent cells.
Highlights
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
Our results have shown that NQO1 activity is considerably increased when HeLa cells reach high density, and this increase correlates with enhanced resistance of cells against H2O2
Cell Cultures—HeLa cells were seeded at a density of 1,500 viable cells/cm2 on 50-cm2 culture dishes in MEM supplemented with 10% fetal calf serum
Summary
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. 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. Expression of the NQO1 gene is positively or negatively regulated by a number of transcription factors (such as c-Jun, Jun-B, Jun-D, c-Fos, Fra, Nrf, and Nrf2) that bind to several cis-elements of the NQO1 gene promoter, including an antiox-
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