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. 2001 May 8;98(10):5550-5.
doi: 10.1073/pnas.101505898. Epub 2001 May 1.

Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1

R S Arnold  1 , J Shi, E Murad, A M Whalen, C Q Sun, R Polavarapu, S Parthasarathy, J A Petros, J D Lambeth
Affiliations

Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1

R S Arnold et al. Proc Natl Acad Sci U S A. .

Abstract

Nox1, a homologue of gp91phox, the catalytic moiety of the superoxide (O(2)(-))-generating NADPH oxidase of phagocytes, causes increased O(2)(-) generation, increased mitotic rate, cell transformation, and tumorigenicity when expressed in NIH 3T3 fibroblasts. This study explores the role of reactive oxygen species (ROS) in regulating cell growth and transformation by Nox1. H(2)O(2) concentration increased approximately 10-fold in Nox1-expressing cells, compared with <2-fold increase in O(2)(-). When human catalase was expressed in Nox1-expressing cells, H(2)O(2) concentration decreased, and the cells reverted to a normal appearance, the growth rate normalized, and cells no longer produced tumors in athymic mice. A large number of genes, including many related to cell cycle, growth, and cancer (but unrelated to oxidative stress), were expressed in Nox1-expressing cells, and more than 60% of these returned to normal levels on coexpression of catalase. Thus, H(2)O(2) in low concentrations functions as an intracellular signal that triggers a genetic program related to cell growth.

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Figures

Figure 1
Figure 1
Expression of Nox1 increases intracellular H2O2. Cells were incubated with 2 μM dichlorofluorescin diacetate, and fluorescence and cell number were determined by flow cytometry. The dashed line indicates vector control (NEF2) cells, and the solid line indicates cells expressing Nox1 (YA28).
Figure 2
Figure 2
Catalase expression and activity in cells transfected with Nox1 and h-catalase. (A) Catalase expression was determined in the indicated cell lines by Western blotting by using an antibody that recognizes both the human (upper band) and mouse (lower band) catalase. Caco-2 cells serve as a positive control for expression of h-catalase. (B) Catalytic activity (100% = 6.5 units/mg cell protein) was assayed in cell lysates. The mean and SE of 4–25 determinations is shown. (C) Reverse transcription–PCR demonstrates Nox1 expression. G3PDH (glyceraldehyde 3-phosphate dehydrogenase) serves as a loading control.
Figure 3
Figure 3
Effect of Nox1 and catalase on the transformed appearance of NIH 3T3 cells. Cell lines that do not (NEF2) or do (YA28, YA28/Z4, YA28/Z3, ZC-1, ZC-5) express Nox1. Lines that express catalase: ZC-1 and ZC-5. (×100.)
Figure 4
Figure 4
Effect of Nox1 and catalase on proliferation of NIH 3T3 cells. Cell lines are as in Fig. 3. Bars show the range of cell counts of duplicate cultures.
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