Orthovanadate and 2,3-Dimethoxy-1,4-Naphthoquinone Augment Growth Factor-Induced Cell Proliferation and c-fos Gene Expression in 3T3-L1 Cells

Our previous study demonstrated an increase in tyrosine protein phosphorylation and phosphatidylinositol phosphorylation induced by oxidants, suggesting a proliferative potential for these reactive substances. In the present study, we focus our investigation on the similarity between a redox cycling naphthoquinone and orthovanadate (VO), an oxidant generator as well as a potent phosphotyrosyl phosphatase inhibitor, in growth of 3T3-L1 cells cultured in serum-free media. Vanadate increased [3H]thymidine incorporation in a concentration-dependent manner. However, in the presence of insulin, epidermal growth factor, or platelet-derived growth factor, VO synergistically augmented, by as much as fivefold, DNA synthesis stimulated by these growth factors. An increase in the association of PI 3-kinase with the insulin receptor was also observed in cells treated with insulin + VO. Expression of the c-fos gene, a marker of early nuclear event in mitogenesis induced by insulin, EGF, and PDGF, but not vasopressin, a non-tyrosine kinase-linked mitogen, was also enhanced by VO. Flow cytometric analysis indicated an acceleration of cell cycle progression when VO and insulin were present simultaneously. Striking similarity was observed between VO and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) in PI 3-kinase activation, c-fos proto-oncogene expression, and DNA synthesis, key events associated with cell growth. Additionally, both VO and DMNQ gave rise to DMPO-.OH adduct EPR signal measured with cell suspensions.These data support an oxidant-mediated increase in tyrosine protein phosphorylation early in the signal transduction cascade of growth factor receptors, leading to augmentation of cell proliferation.