The Effect of Leucine-Rich Alpha-2-Glycoprotein-1 on Cell Survival During Stress

Abstract

Leucine-rich alpha-2-glycoprotein-1 (LRG) is a Cytochrome c (Cyt c) ligand whose function is unknown. It has been hypothesized that LRG may play a role in cell survival by interfering with the apoptogenic function of Cyt c. In order to examine the influence of LRG on cell survival, we transfected MCF-7 breast adenocarcinoma cells with the lrg gene and studied the effect of LRG overexpression on oxidant-induced cell death induced by hydrogen peroxide and DNA damage induced by camptothecin (CPT). In addition, LRG expression was downregulated using short hairpin RNA constructs. Cell survival was measured by the uptake and cleavage of fluorescein diacetate by live cells yielding a fluorescent product. Compared to non-transfected cells as well as cells transfected with the neomycin vector, lrg transfected cells were approximately 3-fold less sensitive to cellular death induced by hydrogen peroxide, an effect comparable to that observed in HL-60 cells for overexpression of the anti-apoptotic protein Bcl-2. In addition, cells with reduced expression of LRG were more sensitive to apoptosis induced by hydrogen peroxide. Upon exposure to CPT, lrg transfected cells demonstrated little, if any, enhanced survival compared to control cells. However, cells with reduced expression of LRG showed greater sensitivity to cell death induced by CPT that was most significant after 72 hours. Our results indicate that LRG plays a role in cell survival in response to both oxidative stress and DNA damaging agents. The mechanism for cell survival may involve inhibition of Cyt c activation of Apaf-1.