ASCORBATE AND ALPHA-TOCOPHEROL PREVENT APOPTOSIS INDUCED BY SERUM REMOVAL INDEPENDENT OF BCL-2

Cells require serum to maintain growth in vitro. Serum provides growth and survival factors and its removal causes an oxidative stress that induces peroxidations in membrane lipids and development of programmed cell death (apoptosis) in some cells, Cells containing Bcl-2 are part ially protected against both lipid peroxidation and apoptosis and some cell lines, such as Daudi, which lack this protein, are very sensitiv e to serum removal. Thus, cells are grown for 48 h in the absence of f etal calf serum and apoptotic cells are scored. HL-60 cells containing a moderate amount of Bcl-2 show 30% apoptosis, while 55% cells are ap optotic of the Bcl-2-negative Daudi cell population. Apoptosis is redu ced to 15% in the transiently transfected Daudi/Bcl-2 cells. Ascorbate (Asc) and alpha-tocopherol (alpha TOH) can prevent lipid peroxidation and apoptosis caused by serum withdrawal, when added to culture media , even in the absence of Bcl-2, Also, these two antioxidants increase survival of cells grown in the absence of serum independent of their B cl-2 content. Immunostaining and quantification of Bcl-2 show that HL- 60 cell line is a heterogeneous population relative to the expression of Bcl-2. When these cells are grown in the presence of serum, cells l acking Bcl-2 survive, but no Bcl-2-negative cells survive without seru m. Part of this population of BCl-2-negative cells is rescued by Asc a nd alpha TOH. Antioxidants effective at the plasma membrane such as As c and alpha TOH can protect cells from oxidative damage and prevent ap optosis independent of Bcl-2 content. (C) 1997 Academic Press.