Puromycin aminonucleoside induces apoptosis and increases HNE in cultured glomerular epithelial cells

Puromycin aminonucleoside induces apoptosis and increases 4-hydroxy-2-nonenal (HNE) in cultured glomerular epithelial cells. We have previously reported the detachment of cultured glomerular epithelial cells (GECs) from their substrata by puromycin aminonucleoside (PAN) treatment. In this study we explored whether or not apoptosis was involved in the mechanisms of the detachment. DNA fragmentation on gel electrophoresis was clearly shown by 10−3 M PAN treatment of GECs. Nuclear staining by Hoechst 33342 indicated the greatest number of apoptotic cells at 10−3 M PAN for 48 h treatment. Similarly, TUNEL methods revealed maximal apoptotic cells at 10−3 M PAN for 48 h treatment. Caspase-3 (like) protease activity increased at 10−3 M PAN, and decreased at 2 × 10−3 M PAN for 48 h treatment as well as at 10−3 M PAN for 60 h treatment. Pretreatment with 2′-deoxycoformycin (DCF), inhibitor of adenosine deaminase, abolished these effects of PAN on cultured GECs. PAN treatment increased HNE, a lipid peroxide adduct, modified protein in cultured GECs, which was also prevented by pretreatment by DCF. These results for the first time indicate that the PAN-induced detachment of GECs from culture substrata is mediated at least in part through apoptosis via oxidative stresses by adenosine deaminase activity.