DIRECT EVIDENCE FOR ANTIOXIDANT EFFECT OF BCL-2 IN PC12 RAT PHEOCHROMOCYTOMA CELLS

Mock-transfected PC12 rat pheochomocytoma cells and PC12 cells transfe cted with the bcl-2 gene, a gene associated with inhibition of apoptos is, were subjected to oxidative stress by incubation in the presence o f the ate-initiator of lipid peroxyl radicals, 2,2 '-azobis(2,4-dimeth ylvaleronitrile) (AMVN). Extraction and chromatographic analysis by tw o-dimensional TLC of the major phospholipid classes showed no differen ces in the phospholipid composition between the mock-and bcl-2-transfe cted cell lines after incubation in the presence of 0.5 mM AMVN for 2 h at 37 degrees C. A method consisting of incorporation of cis-parinar ic acid into the constituent membrane phospholipids before exposure to AMVN was developed to improve the sensitivity of detecting lipid pero xidation in PC12 cells. Analysis of the pattern of changes in parinari c acid-labeled phospholipids after exposure to 0.25 and 0.5 mM AMVN by HPLC showed significant oxidation of phosphatidylcholine (PC), phosph atidylethanolamine (PEA), phosphatidylserine (PS), phosphatidylinosito l (PI), and sphingomyelin (SPH) during a 2-h incubation, The extent of oxidation of each phospholipid class was dependent on the concentrati on of AMVN present up to 1 mM, Based on phospholipid fractional compos ition, the specific rates of PnA peroxidation in phospholipid classes were estimated, In mock-transfected PC12 cells, the order of AMVN-indu ced oxidation effectiveness was the same for both specific rates and r elative rates: PC much greater than PEA > PS > SPH > PI. While a drama tic decrease in both relative and specific oxidation rates was observe d for all phospholipid classes in bcl-2-transfected PC12 cells, the sp ecific oxidation rates were higher for aminophospholipids (PEA and PS) than for other phospholipids. This suggests that antioxidant protecti on by bcl-2-related product(s) may be phospholipid-specific and that a minophospholipids are relatively less protected than the other phospho lipids. The vitamin E analogue, 2,2,5,7,8-pentamethyl-6-hydrochromane, acted as an effective antioxidant in preventing oxidation of parinari c acid-labeled membrane phospholipids during incubation in the presenc e of AMVN and the extent of protection was approximately the same in b oth cell lines. Since, unlike the agents used to generate oxidative st ress in other studies, temperature-driven generation of peroxyl radica ls by AMVN is not dependent on intracellular metabolism, the results p resented provide proof for antioxidant protection, rather than abrogat ion of radical generation afforded by bcl-2 transfection of PC12 cells . (C) 1997 Academic Press.