R. Wen

Attenuation of ischemia-induced mouse brain injury by SAG, a redox-inducible antioxidant protein

Cerebral ischemia resulting from a disruption of blood flow to the brain initiates a cascade of events that causes neuron death and leads to neurologic dysfunction. Reactive oxygen species are thought, at least in part, to mediate this disease process. The authors recently cloned and characterized an antioxidant protein, SAG (sensitive to apoptosis gene), that is redox inducible and protects cells from apoptosis induced by redox agents in a number of in vitro cell model systems. This study reports a neuroprotective role of SAG in ischemia/reperfusion-induced brain injury in an in vivo mouse model. SAG was expressed at a low level in brain tissue and was inducible after middle cerebral artery occlusion with peak expression at 6 to 12 hours. At the cellular level, SAG was mainly expressed in the cytoplasm of neurons and astrocytes, revealed by double immunofluorescence. An injection of recombinant adenoviral vector carrying human SAG into mouse brain produced an overexpression of SAG protein in the injected areas. Transduction of AdCMVSAG (wild-type), but not AdCMVmSAG (mutant), nor the AdCMVlacZ control, protected brain cells from ischemic brain injury, as evidenced by significant reduction of the infarct areas where SAG was highly expressed. The result suggests a rather specific protective role of SAG in the current in vivo model. Mechanistically, SAG overexpression decreased reactive oxygen species production and reduced the number of apoptotic cells in the ischemic areas. Thus, antioxidant SAG appears to protect against reactive oxygen species–induced brain damage in mice. Identification of SAG as a neuroprotective molecule could lead to potential stroke therapies.

Promotion of S‐phase entry and cell growth under serum starvation by SAG/ROC2/Rbx2/Hrt2, an E3 ubiquitin ligase component: Association with inhibition of p27 accumulation

The sensitive‐to‐apoptosis gene (SAG) was initially identified as a redox‐inducible, apoptosis‐protective protein and subsequently found to be the second family member of regulator of cullins (ROC)/RING box protein (Rbx)/Hrt, which acts as a component of E3 ubiquitin ligase. We report here that SAG promoted cell growth under serum starvation. Microinjection of SAG mRNA into quiescent NIH/3T3 cells induced S‐phase entry as determined by [3H]‐thymidine incorporation. Likewise, overexpression of SAG by either adenovirus infection of immortalized human epidermal keratinocytes (Rhek‐1) or DNA transfection of SY5Y human neuroblastoma cells induced cell proliferation under serum starvation. Because cyclin‐dependent kinase inhibitors (CKIs), including p21, p27, and p57, are degraded through the ubiquitin pathway, we tested whether SAG‐induced cell growth is associated with CKI degradation. Although there was no significant difference in the levels of p21 and p57 between the vector controls and SAG‐overexpressing cells, serum starvation induced 10‐ to 18‐fold accumulation of p27 in control Rhek‐1 cells. Accumulation of p27 was remarkably inhibited (only 2 to 5‐fold) in SAG‐infected cells. Inhibition of p27 accumulation was also observed in stably SAG‐overexpressing SY5Y cells. Significantly, SAG‐associated inhibition of p27 accumulation was largely abolished by the treatment with a proteasome inhibitor. In vivo binding of SAG and Skp2, an F‐box protein that promotes p27 ubiquitination, was detected, and the binding was enhanced in SAG‐overexpressing cells grown under serum starvation. Thus, SAG‐induced growth with serum withdrawal appears to be associated with SAG‐mediated p27 degradation. Mol. Carcinog. 30:37–46, 2001.