ABSTRACT
Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1ß foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.
Subject(s)
Cellular Senescence/genetics , DNA Damage , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Reactive Oxygen Species/metabolism , Cell Cycle Checkpoints/genetics , Chromobox Protein Homolog 5 , Humans , Hydrogen Peroxide/metabolism , NADPH Oxidase 4 , NADPH Oxidases/antagonists & inhibitors , Oxidation-ReductionABSTRACT
A 19 KDa heparin binding protein was previously purified from chicken embryos. Essentially localized within basement membranes in early embryonic tissues, this protein is very rich in basic and cystein residues. Its N-terminal fragment is similar to corresponding fragment of two other proteins expressed during embryogenesis and postnatal period. Its synthesis and secretion are induced by retinoic acid in chicken myoblasts and fibroblasts. This new retinoic acid induced heparin binding protein (RI-HB) does stimulate neurite outgrowth and proliferation on PC12 cells. These results suggest that retinoic acid could regulate some aspect of differentiation and development by inducing the synthesis of a new family of growth and neurotrophic factors.
