RRR-ALPHA-TOCOPHERYL SUCCINATE INDUCES APOPTOSIS IN AVIAN RETROVIRUS-TRANSFORMED LYMPHOID-CELLS

The RRR-alpha-tocopheryl succinate form of vitamin E [vitamin E succin ate (VES)] inhibits the proliferation of avian reticuloendotheliosis v irus- transformed RECC- UTC4-1 (C4-1) lymphablastoid cells in a close- dependent manner, blocks the cells in the G2/M cell cycle phase, and i nduces the cells to undergo apoptosis. Apoptosis was documented by dem onstrating changes that are characteristic of this type of cell death, including morphological analyses of chromatin condensation by 4',6-di amidine-2'-phenylindole dihydrochloride (DAPI) staining using scanning confocal and traditional fluorescent microscopy; flow cytometry analy ses of propidium iodide-labeled DNA showing fragmented DNA as a pre-G1 peak; two-color flow cytometry analyses of intact cells labeled first by the TUNEL procedure (terminal deoxynucleotidyl transferase-mediate d deoxyuridine triphosphate-biotin nick-end-labeled DNA stained with f lourescein isothiocyanate-labeled avidin) and then by propidium iodide demonstrating fragmented DNA; and electrophoresis of DNA showing a DN A ladder created by inter nucleosomal DNA fragmentation. The percentag e of apoptotic cells was determined by DAPI staining and showed 11%, 2 7%, and 49% of cells to be apoptotic after treatment with 10 mu g/ml V ES for one, two, and three days, respectively. Analyses of mRNA levels of genes that have been implicated in the apoptotic process, namely, bc1-2, c-myc, and c-jun, revealed no change in bc1-2, decreases in c-m yc mRNA levels after 36 hours of treatment, and increases in c-jun mRN A levels within four hours after treatment. Western immunoblotting ana lyses of protein levels for the transcription factors c-Myc and c-Jun showed normal levels of c-Myc at early time points and decreased level s at 24 and 48 hours after treatment. c-Jun increased as early as 6 ho urs after treatment and returned to lower (yet still elevated over con trol) levels by 48 hours. To determine possible functional consequence s of increased c-Jun expression, gel electrophoretic mobility assays w ere conducted that showed increased AP-1 binding at 24 and 48 hours af ter treatment. These data show that VES induces apoptosis in reticuloe ndotheliosis virus-transformed lymphoid cells and suggest that decreas es of c-Myc protein and increases of c-Jun protein and DNA binding cap acity may be playing a role in VES-mediated events leading to apoptosi s in this cell type.